Hypothalamus-specific polypeptides

ABSTRACT

Disclosed are hypocretin polynucleotides and hypocretin polypeplides, as well as antibodies, oligonucleotides, diagnostic kits and methods, and therapeutic compositions and methods. Hypocretin, one of several novel liypothalamic-specific polypeptides identified isolated and sequenced, is localized to regions of the hypothalamus involved in appetite and feeding behavior. Hypocretin polypeptides are biologically active, producing electrical changes in neurons, lowering body temperature and reducing food intake.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/023,220, filed Aug. 2, 1996, which is explicitly incorporated byreference, as are all references cited herein.

GOVERNMENTAL RIGHTS

This invention was made with governmental support from the United StatesGovernment, National Institutes of Health, Grants GM32355 and NS33396;the United States Government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to the identification, isolation, sequencing,use, and expression of hypothalamus-specific proteins and fragmentsthereof.

BACKGROUND OF THE INVENTION

The hypothalamus, a phylogenetically ancient region of the mammalianbrain, is responsible for the integration of the central nervous systemand the endocrine system and is particularly related to thephysiological response to stress. In contrast to laminar corticalstructures such as the cerebellum and hippocampus whose final functionsrely on innervation from the thalamus and brain stem, the hypothalamusis organized as a collection of distinct, autonomously active nucleiwith discrete functions. Ablation and electrical stimulation studies andmedical malfunctions have implicated several of these nuclei as centralregulatory centers for major autonomic and endocrine homeostatic systemsmediating processes such as reproduction, lactation, fluid balance,metabolism, and aspects of behaviors, such as circadian rhythmicity,basic emotions, feeding and drinking, mating activities, and responsesto stress, as well as normal development of the immune system (Shepherd,G. M., Neurobiology, 3rd ed. Oxford University Press, New York, 1994).Distinct hormones and releasing factors have been associated with someof these nuclei but, at best, the organizations and molecular operationsof these structures are only partially understood.

A substantial portion of a mammal's genetic endowment is dedicated tothe function of its central nervous system, as evidenced by thesubstantial number of mRNAs selectively expressed in the brain(Sutcliffe, J. G., Ann. Rev. Neurosci. 11:157-198, 1988). Many of thesehave been observed to be selectively associated with distinct neuralsubsets. Existing knowledge of the expression of specific hypothalamichormones and releasing factors suggests that ensembles of mRNAsselectively associated with discrete hypothalamic nuclei may encodeproteins singularly associated with the unique functions of thosenuclei.

SUMMARY OF THE INVENTION

The present invention provides peptides and polypeptides found in thehypothalamus region of the mammalian brain. Preferably, the peptides andpolypeptides are enriched in the hypothalamus relative to other regionsof the brain. More preferably the peptides and polypeptides are specificto the hypothalamus. One embodiment is the rat polypeptide hypocretinalso referred to as, H35 protein or clone 35 protein (SEQ ID NO:1) andpolypeptide analogs thereof having at least one conservative amino acidsubstitution. Another embodiment is the mouse hypocretin polypeptide(SEQ ID NO:2) and polypeptide analogs thereof having at least oneconservative amino acid substitution.

The present invention also provides polynucleotides encoding peptidesand polypeptides found in the hypothalamus region of the brain.Preferably, the polynucleotides encoding peptides and polypeptides areenriched in the hypothalamus relative to other regions of the brain.More preferably the polynucleotides encoding peptides and polypeptidesare specific to the hypothalamus. One embodiment is a polynucleotidechosen from the group consisting of the polynucleotide of SEQ ID NO:3, apolynucleotide having at least about 95% of its nucleotide sequenceidentical to the polynucleotide of SEQ ID NO:3, and polynucleotideshybridizing to the polynucleotide of SEQ ID NO: 3. Another embodiment isa polynucleotide chosen from the group consisting of the polynucleotideof SEQ ID NO:4, a polynucleotide having at least about 95% of itsnucleotide sequence identical to the polynucleotide of SEQ ID NO:4, andpolynucleotides hybridizing to the polynucleotide of SEQ ID NO: 4.

Also provided are vectors for the expression of the novelpolynucleotides operably linked to control sequences capable ofdirecting the production of the novel polypeptides in suitable hostcells.

In other aspects this invention provides pharmaceutical compositions ofthe polynucleotides, polypeptides and peptides, antibodies to thepeptides and polypeptides as well as compositions thereof. Thisinvention also provides assay methods and kits for practicing themethods, and methods for using the polynucleotides, peptides andpolypeptides for diagnostic and therapeutic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

In the Drawings,

FIGS. 1A-1C shows the results of subtractive screening, enriched forsequences selectively expressed in hypothalamus. Replicate dot blots onwhich the indicated masses of plasmid DNA for clones of neuron-specificenolase (NSE), cyclophilin, proopiomelanocortin (POMC), vasopressin, thevector pT7T3D, protein kinase Cδ (PKCδ) and growth hormone (GH) weremanually spotted and hybridized with cDNA probes made from cRNAtranscribed from the target or subtracted libraries, or an equal mixtureof the cerebellum and hippocampus driver libraries. Comparison of thesignal intensities for the vasopressin dilution series dots at severallevels of autoradiographic exposure suggested a 20-to-30 fold increasein the specific activity of vasopressin cDNA.

FIGS. 2A-2D shows the results of cDNA library Southern blotting withclones representative of the four distribution classes. Theelectrophoretic lanes contain the cerebellum first driver library (D1),the hippocampus second driver library (D2), and the hypothalamus targetlibrary (T) cleaved with HaeIII and hybridized with the inserts fromclone 35 (Panel A), clone 10 (Panel B), clone 86 (Panel C) and clone 19(Panel D).

FIGS. 3A-3D distribution of hypothalamic mRNAs. Northern blots withpoly(A)⁺ RNA isolated from extracts of whole brain, olfactory bulb,cerebral cortex, hippocampus, hypothalamus, thalamus, cerebellum,pituitary, liver, kidney and heart were probed with cDNA inserts fromthe indicated clones. A cyclophilin probe was included in the series asa control for comparable blot loading and RNA integrity. The twohypothalamus samples represent inadvertent mixtures of approximatelyequal parts of hypothalamus and striatum. The expression patterns aregrouped into four classes (A,B,C,D). Only the regions of the blotscontaining the hybridized signal are shown.

FIGS. 4A-4F depict the expression patterns analyzed by in situhybridization, showing coronal sections of rat brains hybridized withsingle stranded RNA probes corresponding to the inserts of A, clone 35;B, clone 6; C, clone 10; D, clone 20; E, clone 29 and F, clone 21.

FIGS. 5A and 5B show a comparison of rat and mouse cDNA and amino acidsequences corresponding to clone 35 and the amino acid sequence of thepeptide hormone secretin. A. The amino acid sequence is listed on thetop line (rat SEQ ID NO: 1/mouse SEQ ID NO: 2), the rat nucleotidesequence (SEQ ID NO: 14) on the second line and the mouse nucleotidesequence (SEQ ID NO: 15) is listed on the third line. Differences innucleotide sequences are indicated by asterisks below each differentbase, amino acid differences are indicated by alternatives (rat/mouse)listed above the encoding triplets. Tandem basic amino acids (putativesites for proteolytic maturation) are indicated in bold italics, as isthe serine residue most likely to represent the end of the secretionsignal. B. Alignment of hcrt1 and hcrt2 amino acid sequences (SEQ ID NO:7 and SEQ ID NO: 9, respectively) with the amino acid sequence ofsecretin (SEQ ID NO: 21). The first 9 amino acid residues of secretinhave been repeated to indicate apparent circular permutation. Theidentities between the hypocretins and members of theglucagon/vasoactive intestinal polypeptide/secretin family(H.-C.Fehmann, R. Goke, B. Goke, Endocrine Reviews, 16, 390 (1995)) areindicated by asterisks; the hcrt1 and hcrt2 consensus residues (RL LLGNHAAGILT G; SEQ ID NO: 20) appear above the alignment.

FIGS. 6A-6C show the cDNA (SEQ ID NO: 5) and amino acid sequence (SEQ IDNO: 29) of clone 29.

FIGS. 7A-7B are graphical representation of the results of voltage clampexperiments on isolated in vitro rat hypothalamic cells, in whichapplication of 1 μg hcrt2 produced electrical responses in adult but notimmature neurons.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein. All patents and other publications mentioned in thisspecification are expressly incorporated by reference herein.

A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. The standardpolypeptide nomenclature (described in J. Biol. Chem., 243:3552-59(1969) and adopted at 37 CFR §1.822(b)(2)) that provides one letter andthree letter codes for amino acid residues is used.

It should be noted that all amino acid residue sequences representedherein by formulae have a left- to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. In addition, the phrase“amino acid residue” is broadly defined to include modified and unusualamino acids, such as those listed in 37 CFR 1.822(b)(4), andincorporated herein by reference. Furthermore, it should be noted that adash at the beginning or end of an amino acid residue sequence indicatesa peptide bond to a further sequence of one or more amino acid residuesor a covalent bond to an amino-terminal group such as NH₂ or acetyl orto a carboxy-terminal group such as COOH.

Recombinant DNA molecule: a DNA molecule produced by operatively linkingtwo DNA segments. Thus, a recombinant DNA molecule is a hybrid DNAmolecule comprising at least two nucleotide sequences not normally foundtogether in nature.

Receptor: A receptor is a molecule, such as a protein, glycoprotein andthe like, that can specifically (non-randomly) bind to another molecule.

Antibody: The term antibody in its various grammatical forms is usedherein to refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantibody combining site or paratope. Exemplary antibody molecules areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules and portions of an immunoglobulin molecule, including thoseportions known in the art as Fab, Fab′, and F(ab′)₂.

Antibody Combining Site: An antibody combining site is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) an antigen. The term immunoreact in its various forms meansspecific binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3′- to 5′-directionalong the non-coding strand of the DNA or 5′- to 3′-direction along theRNA transcript.

Polypeptide: A linear series of amino acid residues connected to oneanother by peptide bonds between the alpha-amino group and carboxy groupof contiguous amino acid residues.

Protein: A linear series of more than 50 amino acid residues connectedone to the other as in a polypeptide.

Substantially Purified or Isolated: When used in the context ofpolypeptides or proteins, the terms describe those molecules that havebeen separated from components that naturally accompany them. Typically,a monomeric protein is substantially pure when at least about 60% to 75%of a sample exhibits a single polypeptide backbone. Minor variants orchemical modifications typically share the same polypeptide sequence. Asubstantially purified protein will typically comprise over about 85% to90% of a protein sample, more usually about 95%, and preferably will beover about 99% pure. Protein or polypeptide purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a sample, followed byvisualization thereof by staining. For certain purposes, high resolutionis needed and high performance liquid chromatography (HPLC) or a similarmeans for purification utilized.

Synthetic Peptide: A chemically produced chain of amino acid residueslinked together by peptide bonds that is free of naturally occurringproteins and fragments thereof.

Nucleic acid or polynucleotide sequence: includes, but is not limitedto, eucaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNAsequences, comprising the natural nucleoside bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more other bases including, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methyl-inosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methyl-aminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, and 2,6-diaminopurine.

Coding sequence or open reading frame: a polynucleotide or nucleic acidsequence which is transcribed (in the case of DNA) or translated (in thecase of mRNA) into a polypeptide in vitro or in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A transcription termination sequence will usually be located3′ to the coding sequence.

Nucleic acid control sequences: translational start and stop codons,promoter sequences, ribosome binding sites, polyadenylation signals,transcription termination sequences, upstream regulatory domains,enhancers, and the like, as are necessary and sufficient for thetranscription and translation of a given coding sequence in a definedhost cell. Examples of control sequences suitable for eucaryotic cellsare promoters, polyadenylation signals, and enhancers. All of thesecontrol sequences need not be present in a recombinant vector so long asthose necessary and sufficient for the transcription and translation ofthe desired gene are present.

Operably or operatively linked: the configuration of the coding andcontrol sequences so as to perform the desired function. Thus, controlsequences operably linked to a coding sequence are capable of effectingthe expression of the coding sequence. A coding sequence is operablylinked to or under the control of transcriptional regulatory regions ina cell when DNA polymerase will bind the promoter sequence andtranscribe the coding sequence into mRNA that can be translated into theencoded protein. The control sequences need not be contiguous with thecoding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

Heterologous and exogenous: as they relate to nucleic acid sequencessuch as coding sequences and control sequences, denote sequences thatare not normally associated with a region of a recombinant construct,and are not normally associated with a particular cell. Thus, a“heterologous” region of a nucleic acid construct is an identifiablesegment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a construct could includea coding sequence flanked by sequences not found in association with thecoding sequence in nature. Another example of a heterologous codingsequence is a construct where the coding sequence itself is not found innature (e.g., synthetic sequences having codons different from thenative gene). Similarly, a host cell transformed with a construct whichis not normally present in the host cell would be consideredheterologous for purposes of this invention.

Expression system: polynucleotide sequences containing a desired codingsequence and control sequences in operable linkage, so that cellstransformed with these sequences are capable of producing the encodedproduct. In order to effect transformation, the expression system may beincluded on a discrete vector; however, the relevant polynucleotide mayalso be integrated into the host chromosome.

Vector: a recombinant polynucleotide comprised of single strand, doublestrand, circular, or supercoiled DNA or RNA. A typical vector may becomprised of the following elements operatively linked at appropriatedistances for allowing functional gene expression: replication origin,promoter, enhancer, 5′ mRNA leader sequence, ribosomal binding site,nucleic acid cassette, termination and polyadenylation sites, andselectable marker sequences. One or more of these elements may beomitted in specific applications. The nucleic acid cassette can includea restriction site for insertion of the nucleic acid sequence to beexpressed. In a functional vector the nucleic acid cassette contains thenucleic acid sequence to be expressed including translation initiationand termination sites. An intron optionally may be included in theconstruct, preferably ≧100 bp and placed 5′ to the coding sequence.

A vector is constructed so that the particular coding sequence islocated in the vector with the appropriate regulatory sequences, thepositioning and orientation of the coding sequence with respect to thecontrol sequences being such that the coding sequence is transcribedunder the “control” of the control sequences. Modification of thesequences encoding the particular protein of interest may be desirableto achieve this end. For example, in some cases it may be necessary tomodify the sequence so that it may be attached to the control sequenceswith the appropriate orientation; or to maintain the reading frame. Thecontrol sequences and other regulatory sequences may be ligated to thecoding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector whichalready contains the control sequences and an appropriate restrictionsite which is in reading frame with and under regulatory control of thecontrol sequences.

Suitable marker sequences for identification and isolation of correctlytransfected cells include the thymidine kinase (tk), dihydrofolatereductase (DHFR), and aminoglycoside phosphotransferase (APH) genes. Thelatter imparts resistance to the aminoglycoside antibiotics, such askanamycin, neomycin, and geneticin. These, and other marker genes suchas those encoding chloramphenicol acetyltransferase (CAT) andβ-galactosidase (β-gal), may be incorporated into the primary nucleicacid cassette along with the gene expressing the desired therapeuticprotein, or the selection markers may be contained on separate vectorsand cotransfected.

The term “biochemically equivalent variations” means protein or nucleicacid sequences which differ in some respect from the specific sequencesdisclosed herein, but nonetheless exhibit the same or substantially thesame functionality. In the case of cDNA, for example, this means thatmodified sequences which contain other nucleic acids than thosespecifically disclosed are encompassed, provided that the alternate cDNAencodes mRNA which in turn encodes a protein of this invention. Suchmodifications may involve the substitution of only a few nucleic acids,or many. The modifications may involve substitution of degenerate codingsequences or replacement of one coding sequence with another;introduction of non-natural nucleic acids is included. Preferably, themodified nucleic acid sequence hybridizes to and is at least 95%complementary to the sequence of interest.

Similarly, in the case of the proteins and polypeptides of thisinvention, alterations in the amino acid sequence which do not affectfunctionality may be made. Such “biochemically equivalent muteins” mayinvolve replacement of one amino acid with another, use of side chainmodified or non-natural amino acids, and truncation. The skilled artisanwill recognize which sites are most amenable to alteration withoutaffecting the basic function.

The expression products described herein are proteins and polypeptideshaving a defined chemical sequence. However, the precise structuredepends on a number of factors, particularly chemical modificationscommon to proteins. For example, since all proteins contain ionizableamino and carboxyl groups, the protein may be obtained in acidic orbasic salt form, or in neutral form. The primary amino acid sequence maybe derivatized using sugar molecules (glycosylation) or by otherchemical derivatizations involving covalent or ionic attachment with,for example, lipids, phosphate, acetyl groups and the like, oftenoccurring through association with saccharides. These modifications mayoccur in vitro, or in vivo, the latter being performed by a host cellthrough post-translational processing systems. Such modifications mayincrease or decrease the biological activity of the molecule, and suchchemically modified molecules are also intended to come within the scopeof the invention.

B. Hypocretin Proteins and Polypeptides

Hypocretin or clone H35, has been cloned in both rat and mouse. Theamino acid residue sequence in these two mammalian species is notidentical but is sufficiently similar to permit generalization regardingfunction, and so that one can identify and isolate the hypocretin genein any mammalian species.

Variations at both the amino acid and nucleotide sequence level aredescribed in isolates of hypocretin, and such variations are not to beconstrued as limiting. For example, allelic variation within a mammalianspecies can tolerate a several percent difference between isolates of atype of hypocretin, which differences comprise non-deleterious variantamino acid residues. Thus a protein of about 95% homology, andpreferably at least 98% homology, to a disclosed hypocretin isconsidered to be an allelic variant of the disclosed hypocretin, andtherefore is considered to be a hypocretin of this invention.

As disclosed herein, hypocretin is produced first in vivo in precursorform, and is then processed into smaller polypeptides having biologicalactivity as described herein. Insofar as these different polypeptideforms are useful, the term hypocretin protein or polypeptide connotesall species of polypeptide having an amino acid residue sequence derivedfrom the hypocretin gene.

The complete coding nucleotide sequence, clone 35, of rat H35 cDNA is569 nucleotides in length, and is listed in SEQ ID NO 3. The completepreprohypocretin cDNA clone presents a 390 nucleotide open reading frame(ORF) plus triplet termination codons (second line in FIG. 5 and SEQ IDNO. 14). There is a N-terminal signal peptide with a cleavage sitebetween amino acid positions 27 and 28, corresponding to a cleavage siteafter nucleotide position 172 of SEQ ID NO:3.

Translation of this rat cDNA sequence produces a novel protein of 130amino acid residues, referred to as rat preprohypocretin. The amino acidsequence of rat preprohypocretin is listed in SEQ ID NO: 1. The aminoacid sequence of mouse preprohypocretin is listed in SEQ ID NO: 2.

A hypocretin protein of this invention can be in a variety of forms,depending upon the use therefor, as described herein. For example, ahypocretin can be isolated from a natural tissue.

Alternatively, a hypocretin protein of this invention can be arecombinant protein, that is, produced by recombinant DNA methods asdescribed herein. A recombinant hypocretin protein need not necessarilybe substantially pure, or even isolated, to be useful in certainembodiments, although recombinant production methods are a preferredmeans to produce a source for further purification to yield an isolatedor substantially pure receptor composition. A recombinant hypocretinprotein can be present in or on a mammalian cell line or in crudeextracts of a mammalian cell line.

In one embodiment, a hypocretin protein is substantially free of otherneuropeptides, so that the purity of a hypocretin reagent, and thusfreedom from pharmacologically distinct proteins, facilitates use in thescreening methods. The recombinant production methods are ideally suitedto produce significantly improved purity in this regard, althoughbiochemical purification methods from natural sources are also included.In this regard, a hypocretin protein is substantially free from otherneuropeptides if there are insufficient other neuropeptides such thatpharmacological cross-reactivity is not detected in conventionalscreening assays for ligand binding or biological activity.Alternatively, recombinant hypocretin fusion proteins can be produced byjoining nucleotides encoding additional amino acid residue sequence inproper reading frame at the 3′ end of the hypocretin sequence. Thefusion protein thus produced exhibits properties of the added amino acidsequence in addition to the properties of hypocretin. For example, theadditional amino acid sequence may serve to help identify and purity therecombinantly produced hypocretin fusion protein. One preferredhypocretin fusion protein is hypocretin-poly(His).

Preferably, a hypocretin protein of this invention is present in acomposition in an isolated form, i.e., comprising at least about 0.1percent by weight of the total composition, preferably at least 1%, andmore preferably at least about 90%. Particularly preferred is asubstantially pure preparation of hypocretin, that is at least 90% byweight, and more preferably at least 99% by weight. Biochemical methodsuseful for the enrichment and preparation of an isolated hypocretinbased on the chemical properties of a polypeptide are well known, andcan be routinely used for the production of proteins which are enrichedby greater than 99% by weight.

An isolated or recombinant hypocretin protein of this invention can beused for a variety of purposes, as described further herein. Ahypocretin protein can be used as an immunogen to produce antibodiesimmunoreactive with hypocretin. Hypocretin proteins can be used in invitro ligand binding assays for identifying ligand bindingspecificities, and agonists or antagonists thereto, to characterizecandidate pharmaceutical compounds useful for modulating hypocretinfunction, and as therapeutic agents for effecting hypocretin functions.Other uses will be readily apparent to one skilled in the art.

Furthermore, the invention includes analogs of a hypocretin protein ofthis invention. An analog is a man-made variant which exhibits thequalities of a hypocretin of this invention in terms of immunologicalreactivity, ligand binding capacity or the like functional properties ofa hypocretin protein of this invention. An analog can therefore be acleavage product of hypocretin, can be a polypeptide corresponding to aportion of hypocretin, can be hypocretin polypeptide in which a membraneanchor has been removed, and can be a variant hypocretin sequence inwhich some amino acid residues have been altered, to name a fewalternatives.

Insofar as the present disclosure identifies hypocretin from differentmammalian species, the present invention is not to be limited to ahypocretin protein derived from one or a few mammalian species. Thus,the invention includes a mammalian hypocretin protein, which can bederived, by recombinant DNA or biochemical purification from naturalsources, from any of a variety of species including man, mouse, rabbit,rat, dog, cat, sheep, cow, and the like mammalian species, withoutlimitation. Human and agriculturally relevant animal species areparticularly preferred.

Exemplary hypocretin species identified herein are rat and mousehypocretin.

The amino acid reside sequence of rat preprohypocretin is shown in SEQID NO 1, and corresponding nucleotide (cDNA) of rat preprohypocretin isshown in SEQ ID NO 3.

The amino acid residue sequence of mouse preprohypocretin is shown inSEQ ID NO 2, and corresponding nucleotide (cDNA) of mousepreprohypocretin is shown in SEQ ID NO 4.

A hypocretin protein of this invention can be prepared by a variety ofmeans, although expression in a mammalian cell using a recombinant DNAexpression vector is preferred. Exemplary production methods for arecombinant hypocretin are described in the Examples.

The invention also provides a method for the production of isolatedhypocretin proteins, either as intact hypocretin protein, as fusionproteins or as smaller polypeptide fragments of hypocretin. Theproduction method generally involves inducing cells to express ahypocretin protein of this invention, recovering the hypocretin from theresulting cells, and purifying the hypocretin so recovered bybiochemical fractionation methods, using a specific antibody of thisinvention, or other chemical procedures.

The inducing step can comprise inserting a recombinant DNA vectorencoding a hypocretin protein, or fragment thereof, of this invention,which recombinant DNA is capable of expressing a hypocretin, into asuitable host cell, and expressing the vector's hypocretin gene.

As used herein, the phrase “hypocretin polypeptide” refers to apolypeptide having an amino acid residue sequence that comprises anamino acid residue sequence that corresponds, and preferably isidentical, to a portion of a hypocretin of this invention.

A hypocretin polypeptide of this invention is characterized by itsability to immunologically mimic an epitope (antigenic determinant)expressed by a hypocretin of this invention. Such a polypeptide isuseful herein as a component in an inoculum for producing antibodiesthat immunoreact with native hypocretin and as an antigen in immunologicmethods. Representative and preferred hypocretin polypeptides for use asan immunogen in an inoculum are shown herein.

As used herein, the phrase “immunologically mimic” in its variousgrammatical forms refers to the ability of a hypocretin polypeptide ofthis invention to immunoreact with an antibody of the present inventionthat recognizes a conserved native epitope of a hypocretin as definedherein.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of a hypocretin receptor, so long asit includes the required sequence.

In addition, certain hypocretin polypeptides derived from receptorbinding portions of hypocretin have the capacity to inhibit the bindingof the hypocretin that would normally bind a hypocretin receptor. Thus,the invention also includes hypocretin polypeptides which arespecifically designed for their capacity to mimic exposed regions ofhypocretin involved in hypocretin receptor binding interactions andthereby receptor function. Therefore, these polypeptides have thecapacity to function as analogs to hypocretin, and thereby blockfunction.

In addition, polypeptides corresponding to exposed domains have theability to induce antibody molecules that immunoreact with a hypocretinof this invention at portions of hypocretin involved in receptor proteinfunction, and therefor the antibodies are also useful at modulatingnormal hypocretin function.

A hypocretin polypeptide is preferably no more than about 120 amino acidresidues in length for reasons of ease of synthesis. Thus, it morepreferred that a hypocretin polypeptide be no more that about 100 aminoacid residues, still more preferably no more than about 50 residues, andoptimally less than 40 amino acid residues in length when syntheticmethods of production are used. Exemplary polypeptides are hcrt1 andhcrt2.

The present invention also includes a hypocretin polypeptide that has anamino acid residue sequence that corresponds to the sequence of thehypocretin protein shown in the sequence listings, and includes an aminoacid residue sequence represented by a formula selected from the groupconsisting of the polypeptides shown in the sequence listings. In thisembodiment, the polypeptide is further characterized as having theability to mimic a hypocretin epitope and thereby inhibits hypocretinfunction in a classic hypocretin receptor activation assay, as describedherein.

Due to the three dimensional structure of a native folded hypocretinmolecule, the present invention includes that multiple regions ofhypocretin are involved in hypocretin receptor function, which multipleand various regions are defined by the various hypocretin polypeptidesdescribed above. A preferred hypocretin receptor ligand is hcrt. Theability of the above-described polypeptides to inhibit receptor-ligandbinding can readily be measured in a ligand binding assay as is shown inthe Examples herein. Similarly, the ability of the above-describedpolypeptides to inhibit hypocretin receptor function can readily bemeasured in a receptor assay as is described herein.

In another embodiment, the invention includes hypocretin polypeptidecompositions that comprise one or more of the different hypocretinpolypeptides described above which inhibit hypocretin receptor function,admixed in combinations to provide simultaneous inhibition of multiplecontact sites on the hypocretin receptor.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is capable of mimicking an epitope ofhypocretin. Therefore, a present polypeptide can be subject to variouschanges, substitutions, insertions, and deletions where such changesprovide for certain advantages in its use. In this regard, a hypocretinpolypeptide of this invention corresponds to, rather than is identicalto, the sequence of a hypocretin protein where one or more changes aremade and it retains the ability to induce antibodies that immunoreactwith a hypocretin of this invention.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the ability to induceantibody production as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andomithine may be substituted for lysine. D-amino acids may also beincluded in place of one or more L-amino acids. Polypeptides of thepresent invention also include any polypeptide having one or moreadditions and deletions or residues relative to the sequence of apolypeptide whose sequence is shown herein, so long as the requisiteactivity is maintained.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of a hypocretin polypeptide, it is typicallybecause one or more conservative or non-conservative substitutions havebeen made, usually no more than about 30 number percent, more usually nomore than 20 number percent, and preferably no more than 10 numberpercent of the amino acid residues are substituted. Additional residuesmay also be added at either terminus for the purpose of providing a“linker” by which the polypeptides of this invention can be convenientlyaffixed to a label or solid matrix, or carrier. Preferably the linkerresidues do not form a hypocretin epitope, i.e., are not similar isstructure to a hypocretin protein.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not form ahypocretin epitope. Typical amino acid residues used for linking aretyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. Inaddition, a subject polypeptide can differ, unless otherwise specified,from the natural sequence of a hypocretin protein by the sequence beingmodified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolicacid amidation, by terminal-carboxlyamidation, e.g., with ammonia,methylamine, and the like.

When coupled to a carrier to form what is known in the art as acarrier-hapten conjugate, a hypocretin polypeptide of the presentinvention is capable of inducing antibodies that immunoreact withhypocretin. In view of the well established principle of immunologiccross-reactivity, the present invention therefore includes antigenicallyrelated variants of the polypeptides shown herein. An “antigenicallyrelated variant” is a subject polypeptide that is capable of inducingantibody molecules that immunoreact with a polypeptide described hereinand with a hypocretin protein of this invention.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

A hypocretin polypeptide of the present invention, also referred toherein as a subject polypeptide, can be synthesized by any of thetechniques that are known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, ease of production and the like. An excellent summary of themany techniques available can be found in J. M. Steward and J. D. Young,“Solid Phase Peptide Synthesis”, W.H. Freeman Co., San Francisco, 1969;M. Bodansky, et al., “Peptide Synthesis”, John Wiley & Sons, SecondEdition, 1976 and J. Meienhofer, “Hormonal Proteins and Peptides”, Vol.2, p. 46, Academic Press (New York), 1983 for solid phase peptidesynthesis, and E. Schroder and K. Kubke, “The Peptides”, Vol. 1,Academic Press (New York), 1965 for classical solution synthesis, eachof which is incorporated herein by reference. Additional peptidesynthesis methods are described by Sutcliffe in U.S. Pat. Nos. 4,900,811and 5,242,798, which are hereby incorporated by reference. Appropriateprotective groups usable in such synthesis are described in the abovetexts and in J. F. W. McOmie, “Protective Groups in Organic Chemistry”,Plenum Press, New York, 1973, which is incorporated herein by reference.

In general, the solid-phase synthesis methods comprise the sequentialaddition of one or more amino acid residues or suitably protected aminoacid residues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

A hypocretin polypeptide can be used, inter alia, in the diagnosticmethods and systems of the present invention to detect a hypocretinreceptor or hypocretin itself present in a body sample, or can be usedto prepare an inoculum as described herein for the preparation ofantibodies that immunoreact with conserved epitopes on hypocretin.

In addition, certain of the hypocretin polypeptides of this inventioncan be used in the therapeutic methods of the present invention toinhibit hypocretin function as described further herein.

C. Nucleic Acids and Polynucleotides

The DNA segments of the present invention are characterized as includinga DNA sequence that encodes a hypocretin protein of this invention. Thatis, the DNA segments of the present invention are characterized by thepresence of some or all of a hypocretin structural gene. Preferably thegene is present as an uninterrupted linear series of codons where eachcodon codes for an amino acid residue found in the hypocretin protein,i.e., a gene free of introns.

One preferred embodiment is a DNA segment that codes an amino acidresidue sequence that defines a hypocretin protein as defined herein,and the DNA segment is capable of expressing a hypocretin protein ofthis invention. A preferred DNA segment codes for an amino acid residuesequence substantially the same as, and preferably consistingessentially of, an amino acid residue sequence shown in the sequencelisting for a hypocretin protein, such as in SEQ ID NOs 1 and 2.

The amino acid residue sequence of a protein or polypeptide is directlyrelated via the genetic code to the deoxyribonucleic acid (DNA) sequenceof the structural gene that codes for the protein. Thus, a structuralgene or DNA segment can be defined in terms of the amino acid residuesequence, i.e., protein or polypeptide, for which it codes.

An important and well known feature of the genetic code is itsdegeneracy. That is, for most of the amino acids used to make proteins,more than one coding nucleotide triplet (codon) can code for ordesignate a particular amino acid residue. Therefore, a number ofdifferent nucleotide sequences may code for a particular amino acidresidue sequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid residue sequence in all organisms. Occasionally, a methylatedvariant of a purine or pyrimidine may be incorporated into a givennucleotide sequence. However, such methylations do not affect the codingrelationship in any way.

A nucleic acid is any polynucleotide or nucleic acid fragment, whetherit be a polyribonucleotide of polydeoxyribonucleotide, i.e., RNA or DNA,or analogs thereof. In preferred embodiments, a nucleic acid molecule isin the form of a segment of duplex DNA, i.e, a DNA segment, although forcertain molecular biological methodologies, single-stranded DNA or RNAis preferred.

DNA segments (i.e., synthetic oligonucleotides) that encode portions ofhypocretin proteins can easily be synthesized by chemical techniques,for example, the phosphotriester method of Matteucci, et al., (J. Am.Chem. Soc., 103:3185-3191, 1981) or using automated synthesis methods.In addition, larger DNA segments can readily be prepared by well knownmethods, such as synthesis of a group of oligonucleotides that definethe DNA segment, followed by hybridization and ligation ofoligonucleotides to build the complete segment.

Of course, by chemically synthesizing the coding sequence, any desiredmodifications can be made simply by substituting the appropriate basesfor those encoding the native amino acid residue sequence.

Furthermore, DNA segments consisting essentially of structural genesencoding a hypocretin protein can be obtained from recombinant DNAmolecules containing a gene that defines a hypocretin protein of thisinvention, and can be subsequently modified, as by site directedmutagenesis, to introduce any desired substitutions.

1. Cloning Hypocretin Genes

Hypocretin genes of this invention can be cloned by a variety of cloningmethods and from any mammalian species. The cloning is based on theobservation that there is a significant degree of homology betweenmammalian species for any given hypocretin of this invention, andtherefor can be conducted according to the general methods described inthe Examples, using nucleic acid homology strategies.

A typical degree of homology required to successfully clone a hypocretinis at least about 80% homologous at the DNA level, and at least about90% homologous at the protein level. Preferred cloning strategies forisolating a nucleic acid molecule that encodes a hypocretin molecule ofthis invention are described in the Examples, and includes therecitation of polynucleotide probes useful for the screening oflibraries of nucleic acid molecules believed to contain a targethypocretin gene.

Sources of libraries for cloning a hypocretin gene of this invention caninclude genomic DNA or messenger RNA (mRNA) in the form of a cDNAlibrary from a tissue believed to express a hypocretin of thisinvention. Preferred tissues are brain tissues, particularlyhypothalamic tissue. The similarities between rat and mouse hypocretinare further extended to the identification of a sequence of iteration oftrinucleotide CTG repeats. For both mammals, a sequence of fouriterations of the trinucleotide CTG repeats followed by two pairs of CTGare present encoding leucine residues. Thus, the presence of theiterations is typically located within the coding region for the signalpeptide.

Such a triplet expansion in other genes has been implicated as causal inneurological diseases, e.g., myotonic dystrophy as described by Brook etal., Cell, 68:799-808 (1992) and fragile-X syndrome as described by Fuet al., Cell, 67:1047-1058 (1991). In myotonic dystrophy patients whoare mildly affected, at least 50 CTG repeats are present. In severelyaffected individuals, the expansion can exist up to several kilobasepairs. In contrast, in the normal population, the repeat sequence ishighly variable ranging from 5 to 27 copies. Individuals with varyingseverities of fragile-X have been similarly characterized.

Screening for the presence of a region of DNA in which the repeats arepresent in either normal, underexpansion or overexpansion form canprovide a genetic basis for diagnosis for some diseases. The same may betrue for hypocretin in that expansion of the region may contribute tothe basis for a neuronal disorder or disease of the brain or othertissue.

2. Oligonucleotides

The invention also includes oligonucleotides useful for methods todetect the presence of a hypocretin gene or gene transcript (mRNA) in atissue by diagnostic detection methods based on the specificity ofnucleic acid hybridization or primer extension reactions. One embodimentincludes any polynucleotide probe having a sequence of a portion of ahypocretin gene of this invention, or a related and specific sequence.Hybridization probes can be of a variety of lengths from about 10 to5000 nucleotides long, although they will typically be about 20 to 500nucleotides in length. Hybridization methods are extremely well known inthe art and will not be described further here.

In a related embodiment, detection of hypocretin genes can be conductedby primer extension reactions such as the polymerase chain reaction(PCR). To that end, PCR primers are utilized in pairs, as is well known,based on the nucleotide sequence of the gene to be detected.Particularly preferred PCR primers can be derived from any portion of ahypocretin DNA sequence, but are preferentially from regions which arenot conserved in other cellular proteins.

A preferred PCR primer pair useful for detecting hypocretin genes andhypocretin gene expression are described in the Examples. Nucleotideprimers from the corresponding region of hypocretin described herein arereadily prepared and used as PCR primers for detection of the presenceor expression of the corresponding gene in any of a variety of tissues.

3. Expression Vectors

In addition, the invention includes a recombinant DNA molecule(recombinant DNA) containing a DNA segment of this invention encoding ahypocretin protein as described herein. A recombinant DNA can beproduced by operatively linking a vector to a DNA segment of the presentinvention.

The choice of vector to which a DNA segment of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed, these being limitations inherent in the art ofconstructing recombinant DNA molecules. However, a vector of the presentinvention is at least capable of directing the replication, andpreferably also expression, of a hypocretin structural gene included inDNA segments to which it is operatively linked.

In one embodiment, a vector of the present invention includes aprocaryotic replicon, i.e., a DNA sequence having the ability to directautonomous replication and maintenance of the recombinant DNA moleculeextrachromosomally in a procaryotic host cell, such as a bacterial hostcell, transformed therewith. Such replicons are well known in the art.In addition, those embodiments that include a procaryotic replicon alsoinclude a gene whose expression confers drug resistance to a bacterialhost transformed therewith. Typical bacterial drug resistance genes arethose that confer resistance to ampicillin or tetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of a hypocretin gene in a bacterial host cell, such asE. coli, transformed therewith. A promoter is an expression controlelement formed by a DNA sequence that permits binding of RNA polymeraseand transcription to occur. Promoter sequences compatible with bacterialhosts are typically provided in plasmid vectors containing convenientrestriction sites for insertion of a DNA segment of the presentinvention. Typical of such vector plasmids are pUC8, pUC9, pBR322 andpBR329 available from Biorad Laboratories, (Richmond, Calif.), pRSETavailable from Invitrogen (San Diego, Calif.) and pPL and pKK223available from Pharmacia, Piscataway, N.J.

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form therecombinant DNA molecules of the present invention. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), pRc/CMV (Invitrogen, Inc.), the vector pCMV4 described herein,and the like eucaryotic expression vectors.

In preferred embodiments, the eucaryotic cell expression vectors used toconstruct the recombinant DNA molecules of the present invention includea selection marker that is effective in an eucaryotic cell, preferably adrug resistance selection marker. A preferred drug resistance marker isthe gene whose expression results in neomycin resistance, i.e., theneomycin phosphotransferase (neo) gene. Southern et al., J. Mol. Appl.Genet., 1:327-341 (1982). Alternatively, the selectable marker can bepresent on a separate plasmid, and the two vectors are introduced byco-transfection of the host cell, and selected by culturing in theappropriate drug for the selectable marker.

4. Inhibitory Nucleic Acids

In accordance with one embodiment of the invention, nucleic acidmolecules can be used in methodologies for the inhibition of hypocretingene expression, thereby inhibiting the function of thehypocretin:hypocretin receptor binding interaction by blockinghypocretin expression.

To that end, the invention includes isolated nucleic acid molecules,preferably single-stranded nucleic acid molecules (oligonucleotides),having a sequence complementary to a portion of a structural geneencoding a hypocretin protein of this invention. Nucleic acid-basedinhibition is well known and generally referred to as “anti-sense”technology by virtue of the use of nucleotide sequences havingcomplementarily which can hybridize to the “sense” strand or mRNA, andthereby perturb gene expression. Typical oligonucleotides for thispurpose are about 10 to 5,000, preferably about 20-1000, nucleotides inlength and have a sequence capable of hybridizing specifically with astructural protein region of the nucleotide sequence that encodes ahypocretin protein of this invention.

In one embodiment, the invention includes repetitive units of thenucleotide sequence complementary to a portion of a hypocretinstructural gene so as to present multiple sites for complementarybinding to the structural gene. This feature may be provided in a singlenucleic acid segment having repeating sequences defining multipleportions of a structural gene, by physical conjugation of DNA segmentseach containing a single portion of a structural gene, or a combinationthereof comprising conjugates of DNA segments, each having one or moresequences complementary to a structural gene.

Nucleotide base modifications can be made to provide certain advantagesto a DNA segments of this invention, referred to as nucleotide analogs.A nucleotide analog refers to moieties which function similarly tonucleotide sequences in a nucleic acid molecule of this invention butwhich have non-naturally occurring portions. Thus, nucleotide analogscan have altered sugar moieties or inter-sugar linkages. Exemplary arethe phosphorothioate and other sulfur-containing species, analogs havingaltered base units, or other modifications consistent with the spirit ofthis invention.

Preferred modifications include, but are not limited to, the ethyl ormethyl phosphorate modifications disclosed in U.S. Pat. No. 4,469,863and the phosphorothioate modified deoxyribonucleotides described byLaPlanche et al., Nucl. Acids Res., 14:9081, 1986; and Stec et al., J.Am. Chem. Soc., 106:6077, 1984. These modifications provide resistanceto nucleolytic degradation, thereby contributing to the increasedhalf-life in therapeutic modalities. Preferred modifications are themodifications of the 3′-terminus using phosphothioate (PS) sulfurizationmodification described by Stein et al., Nucl. Acids Res., 16:3209, 1988.

In accordance with the methods of this invention in certain preferredembodiments, at least some of the phosphodiester bonds of the nucleotidesequence can be substituted with a structure which functions to enhancethe ability of the compositions to penetrate into the region of cellswhere the hypocretin structural gene to be inhibited is located. It ispreferred that such linkages be sulfur containing as discussed above,such as phosphorotioate bonds. Other substitutions can include alkylphosphothioate bonds, N-alkyl phosphoramidates, phosphorodithioates,alkyl phosphonates, and short chain alkyl or cycloalkyl structures. Inaccordance with other preferred embodiments, the phosphodiester bondsare substituted with structures which are, at once, substantiallynon-ionic and non-chiral.

D. Anti-Hypocretin Antibodies

An antibody of the present invention, i.e., an anti-hypocretin antibody,in one embodiment is characterized as comprising antibody molecules thatimmunoreact with a hypocretin protein of this invention. Preferably, anantibody further immunoreacts with a hypocretin protein in situ, i.e.,in a tissue section.

The invention describes an anti-hypocretin antibody that immunoreactswith any of the hypocretin polypeptides of this invention, preferablyalso immunoreacts with the corresponding recombinant hypocretin protein,and more preferably also reacts with a native protein in situ in atissue section. Preferably, the antibody is substantially free fromimmunoreaction with other proteins or neuropeptides other thanhypocretin. Assays for immunoreaction useful for assessingimmunoreactivity are described herein.

In one embodiment, antibody molecules are described that immunoreactwith a hypocretin receptor polypeptide of the present invention and thathave the capacity to immunoreact with an exposed site on hypocretin thatis required for hypocretin receptor binding. Thus, preferred antibodymolecules in this embodiment also inhibit hypocretin receptor function,and are therefore useful therapeutically to block the receptor'sfunction.

Exemplary hypocretin inhibitory antibodies immunoreact with a hypocretinpolypeptide described herein that defines an exposed region of ahypocretin protein that is involved in hypocretin receptor function,such as ligand binding.

An antibody of the present invention is typically produced by immunizinga mammal with an inoculum containing a hypocretin polypeptide of thisinvention and thereby induce in the mammal antibody molecules havingimmunospecificity for the immunizing polypeptide. The antibody moleculesare then collected from the mammal and isolated to the extent desired bywell known techniques such as, for example, by using DEAE Sephadex toobtain the IgG fraction. Exemplary antibody preparation methods usinghypocretin polypeptides in the immunogen are described herein in theExamples.

The preparation of antibodies against polypeptide is well known in theart. See Staudt et al., J. Exp. Med., 157:687-704 (1983), or theteachings of Sutcliffe, J. G., as described in U.S. Pat. No. 4,900,811,the teachings of which are hereby incorporated by reference.

Briefly, to produce a hypocretin peptide antibody composition of thisinvention, a laboratory mammal is inoculated with an immunologicallyeffective amount of a hypocretin polypeptide, typically as present in avaccine of the present invention. The anti-hypocretin antibody moleculesthereby induced are then collected from the mammal as an antiserum andthose immunospecific for both a hypocretin polypeptide and thecorresponding recombinant hypocretin protein are isolated to the extentdesired by well known techniques such as, for example, by immunoaffinitychromatography. Alternatively, the antiserum may be used.

To enhance the specificity of the antibody, the antibodies arepreferably purified by immunoaffinity chromatography using solidphase-affixed immunizing polypeptide. The antibody is contacted with thesolid phase-affixed immunizing polypeptide for a period of timesufficient for the polypeptide to immunoreact with the antibodymolecules to form a solid phase-affixed immunocomplex. The boundantibodies are separated from the complex by standard techniques.

The word “inoculum” in its various grammatical forms is used herein todescribe a composition containing a hypocretin polypeptide of thisinvention as an active ingredient used for the preparation of antibodiesagainst a hypocretin polypeptide. When a polypeptide is used in aninoculum to induce antibodies it is to be understood that thepolypeptide can be used in various embodiments, e.g., alone or linked toa carrier as a conjugate, or as a polypeptide polymer. However, for easeof expression and in context of a polypeptide inoculum, the variousembodiments of the polypeptides of this invention are collectivelyreferred to herein by the term “polypeptide” and its various grammaticalforms.

For a polypeptide that contains fewer than about 35 amino acid residues,it is preferable to use the peptide bound to a carrier for the purposeof inducing the production of antibodies.

One or more additional amino acid residues can be added to the amino- orcarboxy-termini of the polypeptide to assist in binding the polypeptideto a carrier. Cysteine residues added at the amino- or carboxy-terminiof the polypeptide have been found to be particularly useful for formingconjugates via disulfide bonds. However, other methods well known in theart for preparing conjugates can also be used.

The techniques of polypeptide conjugation or coupling through activatedfunctional groups presently known in the art are particularlyapplicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol.8, Suppl. 7:7-23 (1978) and U.S. Pat. No. 4,493,795, No. 3,791,932 andNo. 3,839,153. In addition, a site-directed coupling reaction can becarried out so that any loss of activity due to polypeptide orientationafter coupling can be minimized. See, for example, Rodwell et al.,Biotech., 3:889-894 (1985), and U.S. Pat. No. 4,671,958.

Exemplary additional linking procedures include the use of Michaeladdition reaction products, di-aldehydes such as glutaraldehyde,Klipstein, et al., J. Infect. Dis., 147:318-326 (1983) and the like, orthe use of carbodiimide technology as in the use of a water-solublecarbodiimide to form amide links to the carrier. Alternatively, theheterobifunctional cross-linker SPDP(N-succinimidyl-3-(2-pyridyldithio)proprionate)) can be used toconjugate peptides, in which a carboxy-terminal cysteine has beenintroduced.

Useful carriers are well known in the art, and are generally proteinsthemselves. Exemplary of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin(BSA) or human serum albumin (HSA), red blood cells such as sheeperythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyaminoacids such as poly D-lysine:D-glutamic acid, and the like.

The choice of carrier is more dependent upon the ultimate use of theinoculum and is based upon criteria not particularly involved in thepresent invention. For example, a carrier that does not generate anuntoward reaction in the particular animal to be inoculated should beselected.

The present inoculum contains an effective, immunogenic amount of apolypeptide of this invention, typically as a conjugate linked to acarrier. The effective amount of polypeptide per unit dose sufficient toinduce an immune response to the immunizing polypeptide depends, amongother things, on the species of animal inoculated, the body weight ofthe animal and the chosen inoculation regimen is well known in the art.Inocula typically contain polypeptide concentrations of about 10micrograms (μg) to about 500 milligrams (mg) per inoculation (dose),preferably about 50 micrograms to about 50 milligrams per dose.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for animals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired immunogenic effect in association with the requireddiluent; i.e., carrier, or vehicle. The specifications for the novelunit dose of an inoculum of this invention are dictated by and aredirectly dependent on (a) the unique characteristics of the activematerial and the particular immunologic effect to be achieved, and (b)the limitations inherent in the art of compounding such active materialfor immunologic use in animals, as disclosed in detail herein, thesebeing features of the present invention.

Inocula are typically prepared from the dried solidpolypeptide-conjugate by dispersing the polypeptide-conjugate in aphysiologically tolerable (acceptable) diluent such as water, saline orphosphate-buffered saline to form an aqueous composition.

Inocula can also include an adjuvant as part of the diluent. Adjuvantssuch as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant(IFA) and alum are materials well known in the art, and are availablecommercially from several sources.

The antibody so produced can be used, inter alia, in the diagnosticmethods and systems of the present invention to detect hypocretinpresent in a sample such as a tissue section or body fluid sample.Anti-hypocretin antibodies that inhibit hypocretin function can also beused in vivo in therapeutic methods as described herein.

A preferred anti-hypocretin antibody is a monoclonal antibody. Apreferred monoclonal antibody of this invention comprises antibodymolecules that immunoreact with a hypocretin polypeptide of the presentinvention as described for the anti-hypocretin antibodies of thisinvention. More preferably, the monoclonal antibody also immunoreactswith recombinantly produced whole hypocretin protein.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature, 256:495-497 (1975), the description of which isincorporated by reference. The hybridoma supernates so prepared can bescreened for the presence of antibody molecules that immunoreact with ahypocretin polypeptide, or for inhibition of hypocretin binding tohypocretin receptor as described herein.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a hypocretin antigen, such as is present in ahypocretin polypeptide of this invention. The polypeptide-inducedhybridoma technology is described by Niman et al., Proc. Natl. Acad.Sci., USA, 80:4949-4953 (1983), the description of which is incorporatedherein by reference.

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 G1X⁺ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention areidentified using the enzyme linked immunosorbent assay (ELISA) describedin the Examples.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that produces and secretes antibody molecules ofthe appropriate polypeptide specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well known techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's Minimal Essential Medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci. USA, 86:5728-5732(1989); and Huse et al., Science, 246:1275-1281 (1989).

The monoclonal antibodies of this invention can be used in the samemanner as disclosed herein for antibodies of the present invention.

For example, the monoclonal antibody can be used in the therapeutic,diagnostic or in vitro methods disclosed herein where immunoreactionwith hypocretin is desired.

Also included in this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention.

E. Diagnostic Methods

The present invention includes various assay methods for determining thepresence, and preferably amount, of hypocretin in a body sample such asa tissue sample, including tissue mass or tissue section, or in abiological fluid sample using a polypeptide, polyclonal antibody ormonoclonal antibody of this invention as an immunochemical reagent toform an immunoreaction product whose amount relates, either directly orindirectly, to the amount of hypocretin in the sample.

Those skilled in the art will understand that there are numerous wellknown clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product whose amount relates to the amount of hypocretinin a body sample. Thus, while exemplary assay methods are describedherein, the invention is not so limited.

For example, in view of the demonstrated property that hypocretin bindsa hypocretin receptor, a hypocretin protein of this invention can beused directly as a probe for detection of a hypocretin receptor bybinding thereto.

Additionally, one can use a nucleic acid molecule probes describedherein to detect the presence in a cell or tissue of a hypocretin geneor expressed gene in the form of mRNA encoding a hypocretin protein ofthis invention, as described further herein. Suitable probe-based assaysare described by Sutcliffe in U.S. Pat. Nos. 4,900,811 and 5,242,798,the disclosures of which are incorporated by reference.

Various heterogenous and homogeneous protocols, either competitive ornoncompetitive, can be employed in performing an assay method of thisinvention.

For example, one embodiment includes a method for assaying the amount ofhypocretin protein in a sample that utilizes an anti-hypocretin antibodyto immunoreact with hypocretin protein in a sample. In this embodiment,the antibody immunoreacts with hypocretin to form a hypocretin-antibodyimmunoreaction complex, and the complex is detected indicating thepresence of hypocretin in the sample.

An immunoassay method using an anti-hypocretin antibody molecule forassaying the amount of hypocretin in a sample typically comprises thesteps of:

(a) Forming an immunoreaction admixture by admixing (contacting) asample with an anti-hypocretin antibody of the present invention,preferably a monoclonal antibody. The sample is typically in the form ofa fixed tissue section in a solid phase such that the immunoreactionadmixture has both a liquid phase and a solid phase, and the antibodyfunctions as a detection reagent for the presence of hypocretin in thesample.

Preferably, the sample is a brain tissue sample that has been preparedfor immunohistological staining as is well known, although other tissuesamples may be adsorbed onto a solid phase, including tissue extracts orbody fluid. In that case the adsorption onto a solid phase can beconducted as described for well known Western blot procedures.

(b) The immunoreaction admixture is maintained under biological assayconditions for a predetermined time period such as about 10 minutes toabout 16-20 hours at a temperature of about 4 degree Celsius to about 45degree Celsius that, such time being sufficient for the hypocretinpresent in the sample to immunoreact with (immunologically bind) theantibody and form a hypocretin-containing immunoreaction product(immunocomplex).

Biological assay conditions are those that maintain the biologicalactivity of the immunochemical reagents of this invention and thehypocretin sought to be assayed. Those conditions include a temperaturerange of about 4 degree Celsius to about 45 degree Celsius, a pH valuerange of about 5 to about 9 and an ionic strength varying from that ofdistilled water to that of about one molar sodium chloride. Methods foroptimizing such conditions are well known in the art.

(c) The presence, and preferably amount, of hypocretin-containingimmunoreaction product that formed in step (b) is determined (detected),thereby determining the amount of hypocretin present in the sample.

Determining the presence or amount of the immunoreaction product, eitherdirectly or indirectly, can be accomplished by assay techniques wellknown in the art, and typically depend on the type of indicating meansused.

Preferably, the determining of step (c) comprises the steps of:

(i) admixing the hypocretin-containing immunoreaction product with asecond antibody to form a second (detecting) immunoreaction admixture,said second antibody molecule having the capacity to immunoreact withthe first antibody (primary) in the immunoreaction product.

Antibodies useful as the second antibody include polyclonal ormonoclonal antibody preparations raised against the primary antibody.

(ii) maintaining said second immunoreaction admixture for a time periodsufficient for said second antibody to complex with the immunoreactionproduct and form a second immunoreaction product, and

(iii) determining the amount of second antibody present in the secondimmunoreaction product and thereby the amount of immunoreaction productformed in step (c).

In one embodiment, the second antibody is a labeled antibody (i.e.,detecting antibody) such that the label provides an indicating means todetect the presence of the second immunoreaction product formed. Thelabel is measured in the second immunoreaction product, therebyindicating the presence, and preferably amount, of second antibody inthe solid phase.

Alternatively, the amount of second antibody can be determined bypreparation of an additional reaction admixture having an indicatingmeans that specifically reacts with (binds to) the second antibody, asis well known. Exemplary are third immunoreaction admixtures with alabeled anti-immunoglobulin antibody molecule specific for the secondantibody. After third immunoreaction, the formed third immunoreactionproduct is detected through the presence of the label.

Exemplary methods involve the use of in situ immunoreaction methodsusing tissue sections, or Western blot procedures, as described bySutcliffe in U.S. Pat. No. 4,900,811.

Another embodiment is a method for assaying the amount oftherapeutically administered hypocretin protein or anti-hypocretinantibody in a body fluid sample such as cerebrospinal fluid (CSF),blood, plasma or serum. The method utilizes a competition reaction inwhich either a hypocretin polypeptide or an anti-hypocretin antibodymolecule of this invention is present in the solid phase as animmobilized immunochemical reagent, and the other of the two reagents ispresent in solution in the liquid phase, in the form of a labeledreagent. A fluid sample is admixed thereto to form a competitionimmunoreaction admixture, and the resulting amount of label in the solidphase is proportional, either directly or indirectly, to the amount ofhypocretin polypeptide or antibody in the fluid sample, depending uponthe format.

One version of this embodiment comprises the steps of:

(a) Forming a competition immunoreaction admixture by admixing(contacting) a fluid sample with:

(1) an anti-hypocretin antibody according to this invention containingantibody molecules that immunoreact with a hypocretin protein of thisinvention, said antibody being operatively linked to a solid matrix suchthat the competition immunoreaction admixture has both a liquid phaseand a solid phase, and

(2) a polypeptide or recombinant hypocretin protein of the presentinvention that is immunoreactive with the added antibody. The admixedpolypeptide/protein in the liquid phase (labeled competing antigen) isoperatively linked to an indicating means as described herein.

(b) The competition immunoreaction admixture is then maintained for atime period sufficient for the competing antigen and the body sampleantigen present in the liquid phase to compete for immunoreaction withthe solid phase antibody. Such immunoreaction conditions are previouslydescribed, and result in the formation of an indicating means-containingimmunoreaction product comprising the labeled competing antigen in thesolid phase.

(c) The amount of indicating means present in the product formed in step(b) is then determined, thereby determining the presence, and preferablyamount, of sample antigen present in the fluid sample.

Determining the indicating means in the solid phase is then conducted bythe standard methods described herein.

A reverse version of this embodiment comprises the steps of:

(a) Forming a competition immunoreaction admixture by admixing a fluidsample with:

(1) an anti-hypocretin antibody according to the present invention; and

(2) a hypocretin polypeptide or recombinant hypocretin protein of thepresent invention (capture antigen) that is immunoreactive with theantibody and is operatively linked to a solid matrix such that thecompetition immunoreaction admixture has both a liquid phase and a solidphase.

(b) The competition immunoreaction admixture is then maintained for atime period sufficient for any hypocretin antigen or anti-hypocretinantibody in the fluid to compete with the admixed antibody molecules forimmunoreaction with the solid phase capture antigen and form anantibody-containing immunoreaction product in the solid phase.

(c) The amount of antibody present in the product formed in step (b) isthen determined, thereby determining the presence and amount of targetmaterial in the fluid sample.

In preferred embodiments, the antibody is operatively linked to anindicating means such that the determining in step (c) comprisesdetermining the amount of indicating means present in the product formedin step (b).

Preferably, the fluid sample is provided to a competition immunoreactionadmixture as a known amount of CSF, blood, or a blood derived productsuch as serum or plasma. Further preferred are embodiments wherein theamount of immunochemical reagent in the liquid phase of theimmunoreaction admixture is an excess amount relative to the amount ofreagent in the solid phase. Typically, a parallel set of competitionimmunoreactions are established using a known amount of purifiedrecombinant hypocretin or polypeptide in a dilution series so that astandard curve can be developed, as is well known. Thus, the amount ofproduct formed in step (c) when using a fluid sample is compared to thestandard curve, thereby determining the amount of target antigen presentin the fluid.

In another embodiment, the method for assaying the amount of hypocretinin a sample utilizes a first capture antibody to capture and immobilizehypocretin in the solid phase and a second indicator antibody toindicate the presence of the captured hypocretin antigen. In thisembodiment, one antibody immunoreacts with a hypocretin protein to forma hypocretin-antibody immunoreaction complex, and the other antibody isable to immunoreact with the hypocretin while present in thehypocretin-antibody immunoreaction complex. This embodiment can bepracticed in two formats with the immobilized capture antibody beingeither of the two above-identified antibodies, and the indicatorantibody being the other of the two antibodies.

Where an antibody is in the solid phase as a capture reagent, apreferred means for determining the amount of solid phase reactionproduct is by the use of a labeled hypocretin polypeptide, followed bythe detection means described herein for other labeled products in thesolid phase.

Also included are immunological assays capable of detecting the presenceof immunoreaction product formation without the use of a label. Suchmethods employ a “detection means”, which means are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelpolypeptides, methods and systems. Exemplary detection means includemethods known as biosensors and include biosensing methods based ondetecting changes in the reflectivity of a surface, changes in theabsorption of an evanescent wave by optical fibers or changes in thepropagation of surface acoustical waves.

Alternative methods of expression, amplification, and purification willbe apparent to the skilled artisan. Representative methods are disclosedin Sambrook, Fritsch, and Maniatis, eds. Molecular Cloning, a LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory (1989) and in Ausabel etal., eds., Current Protocols in Molecular Biology, Wiley & Sons, Inc.,New York (1989).

D. Specific Methods

Directional tag PCR subtractive hybridization was used to enrich a cDNAlibrary for clones of mRNA species selectively expressed in thehypothalamus. Candidate clones identified by their hybridization to asubtracted hypothalamus probe were validated in three stages. First, ahigh throughput cDNA library Southern blot was used to demonstrate thatthe candidate corresponded to a species enriched in the subtractedlibrary. Second, candidate clones positive in the first assay were usedas probes for Northern blots with RNA from several brain regions andperipheral tissues. Finally, candidate clones that were still positivewere subjected to in situ hybridization analysis to detect thehypothalamic regions that express the corresponding mRNAs.

Typically, subtractive hybridization protocols utilize a singletarget-driver dichotomy for enrichment of target-specific species. Inthe current study, a two-step subtraction protocol, first depletinghypothalamus sequences with a cerebellum driver, and then with ahippocampus driver, was employed. Previous studies using single stepsubtraction methodology had been successful in finding clones of speciesenriched in a target compared to the single driver tissue, only to findconsiderable expression in other brain regions. The present protocol wasdesigned to provide a more stringent selection for clones of mRNAs withhigh selectivity for the target. Grids of the subtracted library wereprepared and probed as described by Usui, H., Falk, J. D., Dopazo, A.,de Lecea, L., Erlander, M. G., & Sutcliffe, J. G., J. Neurosci.14:4915-4926 (1994). DNA sequence analysis, Northern blotting and insitu hybridization were performed as described by Usui et al., supra,and de Lecea, L., Soriano, E., Criado, J. R., Steffensen, S. C.,Henriksen, S. J., & Sutcliffe, J. G., Molec. Brain Res 25:286-296(1994).

In situ hybridization analysis was performed essentially as described byGall, C. M. & Isackson, P. M., Science 245: 758-761 (1989) and byErlander, M. G., et al., Proc. Natl. Acad. Sci. USA 90: 3452-3456(1993). Coronal sections about 25 μm thick cut from brains of adultSprague-Dawley rats were hybridized at 55 degrees Celsius for 16 hourswith ³⁵S-labelled single-stranded RNA probes at 10⁷ counts per minuteper ml. Free-floating sections were treated with Rnase A at 4 μg/ml at37 degrees Celsius for 1 hour and washed in 1×SSC (15 mM NaCl, 1.5 mM Nacitrate), 50% formamide at 55 degrees Celsius for 2 hours. Finalstringency washes were in 0.1×SSC at 68 degrees Celsius for 1 hour.Sections were mounted on coated slides, dehydrated and exposed to KodakXAR film for 5 days at room temperature.

For cDNA library Southern blotting, 2 μg of each library was digestedwith HaeIII, separated by electrophoresis, transferred to nylonmembranes, and hybridized to individual clones, as described in Usui etal, supra.

To recognize mRNAs that are selectively expressed in the hypothalamus,poly(A)-enriched cytoplasmic RNA from carefully dissected rat and mousehypothalami were prepared. Target cDNA libraries in vector pT7T3D(Pharmacia Biotech, Piscataway, N.J.) and driver libraries inpGEM11Zf(−) (Promega, Madison, Wis.) from analogously preparedcerebellar and hippocampal RNA samples were constructed. The directionaltag PCR subtractive hybridization method of Usui and colleagues in Usuiet al., supra was applied to produce tagged hypothalamic cDNAs fromwhich cerebellar and hippocampal sequences were depleted in twoconsecutive steps, removing more than 97% of the input target cDNA. Thetag sequences were used as PCR primer-binding sites to amplify theremaining material. An aliquot of the amplified product was cloned intopBCSK⁺ (Stratagene, La Jolla, Calif.) to generate a subtractedhypothalamus library with 5×10⁵ members, with inserts ranging from 400to 1200 (average 700) nucleotide pairs, as judged by agarose gelelectrophoresis of the released inserts.

To validate the efficiency of the subtraction, the degree of depletionin the subtracted library of sequences known to be expressed panneurallyand the enrichment of sequences known to be expressed specifically inthe hypothalamus was determined. Dot blots were prepared with dilutionsof cDNA clones of the mRNAs encoding the following proteins: panneuralneuron-specific enolase, ubiquitously expressed cyclophilin,hypothalamus-specific vasopressin, hypothalamus-enrichedproopiomelanocortin (POMC), thalamus-specific protein kinase C6, andpituitary-specific growth hormone, as well as the target vector itself.The blots were probed with cDNA inserts amplified by PCR from theunsubtracted target library, the subtracted target library or a pool ofthe driver libraries (FIG. 1). The driver and unsubtracted-libraryprobes gave strong signals for cyclophilin and neuron-specific enolase,and a weaker signal for POMC. Neither hippocampus nor cerebellum isknown to express POMC. Although this finding could be explained if oneof the drivers had suffered contamination with mRNA from anotherstructure, for example brain stem, the studies below suggest that thesignal with the driver libraries was probably due to backgroundhybridization to sequences in the POMC clone. The unsubtracted targetadditionally gave a weak signal for vasopressin. The subtracted probegave a very strong signal for vasopressin and POMC and otherwise onlyfaint or undetectable signals. The increase in strength of thevasopressin signal was 20-to-30 fold. Thus, the subtraction protocolremoved abundant, panneurally expressed sequences nearly quantitativelywhile enriching for hypothalamus-specific sequences. There was noapparent contamination with sequences from the anatomically adjacentstructures, thalamus or pituitary. The effectiveness of the subtractionwas quantitated further by measuring the frequencies of VAT-1 andoxytocin clones in the unsubtracted and subtracted libraries by colonyhybridization with a probe corresponding to a mixture of clones of thesetwo species. The frequency of positive clones in the unsubtracted targetwas 4/2775. After subtraction, the frequency increased to 33/1224. Thesefrequencies indicate an approximately 19-fold increase in the specificactivities of these known hypothalamus-enriched species, consistent withthe estimates suggested by the data of FIG. 1.

To identify species enriched by the subtraction, 648 clones from thesubtracted library were placed into grid arrays and hybridized to threereplicate blots of grid images with probes prepared from theunsubtracted or subtracted target library, or a pool of the driverlibraries. Approximately 70% of the colonies gave significant signalswith the subtracted target probe compared to 50% with the unsubtractedtarget probe. Only 10% of the colonies gave signals with themixed-driver probe.

Plasmid DNA was prepared individually from 100 of the colonies that gavestrong signals with target-derived probes but no signal with themixed-driver probe. Partial sequences of the inserts were determined for94 of these, using a sequencing primer that annealed to the vectorregion adjacent to the 3′ ends of the inserts. The remaining 6 cloneswere not pursued further because clear sequences were not obtained. Morethan 90% of the 3′ sequences appeared to be derived from bona fide 3′ends of mRNAs as they contained recognizable poly(A)-addition consensushexads (Birnstiel, M. L., Busslinger, M., & Strub, K. Cell 41:349-359,1985) 12-22 nucleotides upstream from the poly(A) tracts used in theirdirectional cloning. The sequences were searched by BLAST analysis(Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D., J.Molec. Biol. 215:403, 1990) against the GenBank database. For those thatappeared to be novel, the sequence at the 5′ end of the insert was alsodetermined and compared with the database.

A compilation of those data is presented in Table 1 and databaseaccession numbers are given for those prototypes for which a match wasfound. The 94 clones from the subtracted library for which data wereobtained corresponded to 43 distinct mRNA species. Twenty-nine of thesewere encountered only once in the set of 94 clones, while 14 specieswere seen between 2 to 13 times. Among the 43 distinct species were 21that were unambiguously matched to known mRNA species and 22 that werenovel species. Amongst the novel species were 6 that appear tocorrespond (greater than 80% nucleotide sequence identity across anextensive span) to rat homologues of so-called “expressed sequence tags”(ESTs), mRNAs of as yet unknown function compiled in the databases. Twospecies exhibited similarities in both their partial nucleotidesequences and putative encoded amino acid sequences that suggest them torepresent members of protein families: a protein related to the VAT-1secretory vesicle protein (clone 6), and a new calmodulin-dependentprotein kinase (clone 29, SEQ ID NO:5).

The cDNA insert from at least one representative of each of the 43 mRNAspecies was used as a probe in a Southern blot with lanes correspondingto the hypothalamus, hippocampus and cerebellum target and driver cDNAlibraries, each cleaved with the restriction endonuclease HaeIII.Assuming that the cDNA libraries are representative of the mRNAsexpressed in their corresponding tissues, this assay serves as a lowcost, high throughput surrogate for more expensive and time consumingNorthern blot analyses. The hybridization results of the clones in thisso-called “cDNA library Southern blot” assay were classified in one offive patterns (Table 1): hybridization to bands detected exclusively inthe hypothalamus library (A), to bands highly enriched in hypothalamusbut still detectable in hippocampus and cerebellum lanes (B), to bandsin hypothalamus and hippocampus but not in cerebellum (C), to bands inall three tissues (D), or too faint to categorize (E). Examples ofclasses A-D are shown in FIG. 2. Twenty-three of the 43 distinct mRNAspecies were exclusive to or highly enriched in the hypothalamuslibrary, and an additional 15 species were undetectable in thecerebellum library, indicating the effectiveness of the protocols foridentifying species selectively present in the target library. It may besignificant that the patterns classified as D corresponded to clonesthat were isolated only once; similarly, none of the species lacking apoly(A)-addition signal turned up more than once. The existence in thecollection of species present in hippocampal, but not cerebellar,libraries presumably is explained by their enrichment during the firstsubtraction step with cerebellum driver to an extent that did not allowtheir complete depletion in the second step with hippocampus driver.POMC gave an A pattern in this assay, demonstrating that the driverlibraries were not significantly contaminated with POMC-expressingstructures. Thus the low POMC signal observed with the driver probes inFIG. 1 is mostly likely accounted for by vector cross-hybridization.

Northern blots were performed for 15 of the species that showedhypothalamus-enriched or -specific distributions (group A or B) in thecDNA Southern blot assay. The blots (FIG. 3) included RNA samples from 6grossly dissected regions of rat brain in addition to pituitary, liver,kidney and heart. For the clones of species that had been isolated twoor more times, the correspondence with the cDNA library Southern blotassay was excellent. Thus, clones 2 (oxytocin) and 35 (novel), whichgave A patterns in the cDNA Southern blot study, each detected a bandthat was strong in the hypothalamus lanes, but only very faint orundetectable in the other lanes. The faint signals were possibly due tolow expression in those tissues or to contamination during tissuedissection. Clones 6 (VAT1-like), 10 (novel) and 12 (novel), which hadgiven B patterns, each detected bands that were considerably moreintense in the hypothalamus than hippocampus or cerebellum lanes,although each was detected in the pituitary lane (6 strongly) and in thesamples from some other structures. Clones 3 (novel), 15 (novel) and 29(novel calmodulin-dependent protein kinase), although classifiedoriginally as B patterns, are more properly considered as C patterns, astheir expression profiles in this assay are not enriched in hypothalamusper se, but rather are low in the cerebellum.

The clones encountered only once behaved, as a group, less well. Clones21 (novel), 37 (novel), 98 (novel) and 99 (kinesin) failed to showsubstantial enrichment in hypothalamus over hippocampus or cerebellum(although 98 was thalamus enriched). However, clone 33 (novel) detectedan RNA species more prevalent in hypothalamus and thalamus than cortex,pons or olfactory bulb and was undetectable in hippocampus, cerebellumor peripheral tissues; thus, technically speaking, clone 33 maintainedits A pattern classification. Clone 20 (novel) detected an RNA specieswith ubiquitous expression but enrichment in hypothalamus and thalamus,thus it is more properly classified as B pattern. Clone 67 (novel)detected a species enriched in hypothalamus and olfactory bulb that wasdetectable in other brain regions and pituitary but was not detectablein cerebellum.

In situ hybridization on coronal sections of brain from adult male ratswas performed using the inserts from clones representing all fourclasses (A-D): 6, 10, 20, 21, 29 and 35. For all clones, thehybridization pattern was consistent with the Northern blot data. In theA class, the clone 35 mRNA displayed a striking pattern of bilaterallysymmetric expression restricted to a few cells in the paraventricularhypothalamic area and ependymal cells surrounding the brain ventricles.No clone 35 signals were detected outside the hypothalamus. The sequenceof clone 35 is shown in FIG. 5 and SEQ ID NO. 14.

Clones 6, 10 and 20, belonging to class B, displayed somewhat morecomplex distributions. Clone 6 gave strong signals in theperiventricular hypothalamic nucleus, anterior hypothalamic area,preoptic and arcuate nuclei. Very strong hybridization could also beseen in the centromedial thalamic nucleus and medial habenula. Clone 10displayed almost the same pattern but additional strong signals could beseen in the laterodorsal thalamic nucleus and dentate gyrus, with weaksignals in the hippocampal CA fields and the entire neocortex.Interestingly this mRNA showed a marked enrichment in basal diencephalicstructures that included nuclei not only of the hypothalamus but also ofthe amygdaloid complex. Clone 20 exhibited low levels of expression inseveral areas of the brain, but displayed especially strong signals inthe ventral hypothalamus, most notably in the anterior hypothalamic andperiventricular nuclei.

Clone 29 (class C, SEQ ID NO:5), which encodes a novel calmodulinkinase-like protein, was also very strongly expressed in the anteriorhypothalamic area and arcuate nucleus, as well as in the pyramidal celllayer of all hippocampal fields and in the medial and central nuclei ofthe amygdala. The sequence of clone 29 is shown in FIG. 6. Clone 21represents a class D cDNA, whose distribution includes hypothalamic aswell as extrahypothalamic structures. In particular, the clone 21 mRNAwas found in cortex, amygdala, hippocampus, caudate, and severalthalamic (centrodorsal and reticular nuclei) and hypothalamic nuclei.Within the hypothalamus, clone 21 mRNA was especially abundant in theparaventricular hypothalamic nucleus.

The data compiled in Table 1 suggest that this strategy was effective:53 of the 94 clones studied were shown to correspond to mRNAs expressedin the hypothalamus at much higher concentrations than in either thehippocampus or cerebellum. An additional 32 of the clones were enrichedin both hypothalamus and hippocampus over cerebellum, indicating thatthe first subtraction was more efficient, probably because the targetconcentration was higher in the hybridization reaction, thus a greaterportion of the common species were driven into hybrids. Cumulatively, 85of the 94 candidates were found to be enriched in the targethypothalamus compared to the cerebellum, a quite acceptable successrate. It is noteworthy that in 8 cases, the cDNA library Southern blotassay suggested a higher degree of hypothalamus enrichment than waslater observed by Northern blotting, presumably due to artifactualenrichment in the target libraries compared to the driver libraries. Ina few cases this can be explained by artifactual cloning of an internalor intronic cDNA fragment. Other cases may be explained by difficultiesin achieving proportional representation of low prevalence mRNAs in cDNAlibraries.

The subtraction steps provided an approximately 30-fold enrichment. Inthe secondary screen, approximately 60% of the clones were positive withthe subtracted probe but not the target probe. Of the 94 clones selectedfrom this screen, 53 were clones of mRNAs selectively expressed inhypothalamus. These 53 clones correspond to approximately 1% of theclones examined in this pilot study, and represented 16 distinct mRNAspecies, suggesting that a complete characterization of hypothalamusmRNAs might reveal 100-200 species that were specific to or highlyenriched in the hypothalamus. Of the 16 mRNA species detected here, 9corresponded to already known proteins, among them oxytocin, vasopressinand POMC, three neuropeptides known to be highly enriched in thehypothalamus. However, 7 mRNA species were novel. Among mRNA species notdetected in the 94-clone sample were those encoding the releasingfactors, which are less abundant than most of the species detected here.

Oxytocin and vasopressin mRNAs are predominantly associated withdiscrete hypothalamic nuclei, as was previously known. The in situhybridization images indicate that several additional mRNAs, includingseveral novel species, are enriched in the hypothalamus. Among the novelspecies, only clone 35 meets the hypothesis in its strictest sense: themRNA appears to be restricted to nuclei in the paraventricular area ofthe hypothalamus.

Other mRNAs corresponding to novel clones exhibit enrichment in basaldiencephalic structures, especially the hypothalamus, but within thehypothalamus none is restricted to a single nucleus. These speciespresumably encode proteins whose functions are not dedicated to singlephysiological systems. Nevertheless, their roles seem to have selectiveutility within the CNS. Previous studies looking at mRNAs enriched inthe caudate revealed several involved in signal transduction pathways(Usui, et al., supra). That is not the finding for thehypothalamus-enriched species encountered thus far.

The data suggest that the hypothalamus utilizes at least two differentstrategies for employing selectively expressed proteins. Some specificmRNAs are discretely correlated to distinct nuclei. Thus far, all ofthese mRNAs encode secretory signalling proteins. A class of mRNAs havealso been recognized that are expressed prominently in hypothalamus andamygdala. These do not appear to be restricted to functionally discreteregions, but their comparable anatomical restrictions suggest that theymight participate in a series of biochemical processes that areselectively distributed to these regions, which are developmentallyrelated. Thus these regions may share molecular properties that are notapparent at the anatomical level.

DNA sequence analysis of the complete 569 nucleotide rat clone 35revealed that the clone mRNA encodes a 130-residue putative secretoryprotein (called H35) or hypocretin with 4 sites for potentialproteolytic maturation (FIG. 5 and see SEQ ID NO. 14). Severalproteolytic fragments have been identified, some replacing C-terminalglycines with amide groups. Two of the products of proteolysis have 14amino acid identities across 20 residues. This region of H35 includes a7/7 match with a region of the gut hormone secretin, suggesting that theprepropeptide gives rise to two peptide products that are structurallyrelated both to each other and to secretin.

The mouse homolog of clone 35 was also isolated and sequenced (FIG. 5and see SEQ ID NO. 15). The mouse nucleotide sequence differs in 35positions relative to the rat sequence and contains 16 additionalnucleotides near its 3′ end. Of these differences, 19 nucleotides differwithin the protein coding region. Only 7 of these affect the encodedprotein sequence. One amino acid difference is a neutral substitution inthe secretion signal sequence (residue 3). The remaining 6 differencesare in the C-terminal region. One of these obliterates a potentialproteolytic cleavage site. This observation and the nature of the otherdifferences make it unlikely that 2 of the possible maturation productsof the rat preproprotein are functional. However, the 2 peptides thatare related both to each other and to secretin are absolutely preservedbetween species, providing strong support for the notion that thesepeptides have a function conserved during evolution.

The cells that express this mRNA are distributed in a bilaterallysymmetrical pattern in a previously uncharted nucleus of the ratdorsal-lateral hypothalamus and sparse ependymal cells that line theventricles suggesting that the peptides function as intercellularmessengers within the CNS. Colocalization studies suggest a partialoverlap with cells positive for galanin, bradykinin and dynorphin. Therat H35 mRNA is restricted to the CNS in the studies performed to date.It is not expressed at high concentrations in immature animals.

These observations, along with the sequence data discussed above,suggest that the H35 peptides are secreted into the CSF and locallywithin the hypothalamus; that their functions are only manifested inmature animals; and that their expression is coupled to the generalhomeostatic status of the animal, although not regulated in anall-or-none fashion by homeostasis. In other words, these are newhormones that act within the central nervous system.

The polypeptides may be expressed by transformation of a suitable hostcell with a cDNA in a suitable expression vector. The choice of hostcell is not critical. The polypeptide may be produced from a procaryotic(e.g. E. coli) or eucaryotic (mammalian, e.g. COS-7, CHO, NIH 3T3) hostcell, as desired.

The hypocretin polypeptides, and fragments thereof, of this inventionare useful in diagnosis and therapy. Recombinant or natural polypeptidesmay be used in Western blot, ELISA, RIA, and the like, and in receptorbinding assays, for direct or competitive binding studies to identifyhyprocretin specific receptors. The identification of hypocretin analogsand antagonists is also accomplished via use of the polypeptidesidentified herein. Further details of such uses are described in U.S.Pat. No. 5,242,798, incorporated herein by reference.

In another aspect, the polypeptides of this invention may be used togenerate antibodies. Methods of preparing polyclonal antibodies are wellknown in the art. For example, an immunogenic conjugate comprising thehypocretin protein or a fragment thereof, optionally linked to a carrierprotein, is used to immunize a selected mammal (mouse, rabbit, et al.).Serum from the immunized mammal is collected and treated to separate theimmunoglobulin fraction. Monoclonal antibodies are prepared by standardhybridoma cell technology (Koller and Milstein, Nature 256:495-497(1975)). Briefly, spleen cells are obtained from a host animal immunizedwith an hypocretin protein or fragment. Hybrid cells are formed byfusing these spleen cells with an appropriate myeloma cell line andcultured. The antibodies produced are screened for their ability to bindH35 by, for example, ELISA. The cells producing the hypocretin antibodyare selected.

Antibodies directed to a conserved epitope common to the hypocretinpolypeptides of several species will detect hypocretin polypeptides ofmammalian species in general. For example, antibodies directed againstsuch a conserved sequence as GNHAAGILT (SEQ ID NO: 13; FIG. 5B) can beused to detect human hypocretin polypeptides.

The polynucleotides and polypeptides of this invention may also beformulated into diagnostic and therapeutic compositions. Representativemethods of formulation may be found in Remington: The Science andPractice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa. (1995).The selection of the precise concentration, composition, and deliveryregimen is influenced by, inter alia, the specific pharmacologicalproperties of the selected compound, the intended use, the nature andseverity of the condition being treated or diagnosed, and the physicalcondition and mental acuity of the intended recipient. Suchconsiderations are within the purview of the skilled artisan.

Representative delivery regimens include oral, parenteral (subcutaneous,intramuscular, and intravenous), rectal, buccal, pulmonary, transdermal,and intranasal, preferably intravenous. The composition may be in solid,liquid, gel, or aerosol form. Generally, the compound will be present inan amount from about 1 μg to about 100 μg, in a sterile aqueoussolution, optionally including stabilizers and the like.

The present invention also describes a diagnostic system, preferably inkit form, for assaying for the presence of a hypocretin of thisinvention in a body sample, such brain tissue, cell suspensions ortissue sections, or body fluid samples such as CSF, blood, plasma orserum, where it is desirable to detect the presence, and preferably theamount, of a hypocretin protein in the sample according to thediagnostic methods described herein.

In a related embodiment, a nucleic acid molecule can be used as a probe(an oligonucleotide) to detect the presence of a gene or mRNA in a cellthat is diagnostic for the presence or expression of a hypocretin in thecell. The nucleic acid molecule probes were described in detail earlier.

The diagnostic system includes, in an amount sufficient to perform atleast one assay, a subject hypocretin polypeptide, a subject antibody ormonoclonal antibody, and a subject nucleic acid molecule probe of thepresent invention, as a separately packaged reagent.

Another embodiment is a diagnostic system, preferably in kit form, forassaying for the presence of a hypocretin polypeptide or anti-hypocretinantibody in a body fluid sample such as for monitoring the fate oftherapeutically administered hypocretin polypeptide or anti-hypocretinantibody. The system includes, in an amount sufficient for at least oneassay, a subject hypocretin polypeptide and a subject antibody as aseparately packaged immunochemical reagent.

Instructions for use of the packaged reagent(s) are also typicallyincluded.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic (e.g., polyethylene, polypropylene orpolycarbonate), paper, foil and the like capable of holding within fixedlimits a polypeptide, polyclonal antibody or monoclonal antibody of thepresent invention. Thus, for example, a package can be a glass vial usedto contain milligram quantities of a hypocretin polypeptide or antibodyor it can be a microliter plate well to which microgram quantities of acontemplated polypeptide or antibody have been operatively affixed,i.e., linked so as to be capable of being immunologically bound by anantibody or antigen, respectively.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent or sample admixtures,temperature, buffer conditions and the like.

A diagnostic system of the present invention preferably also includes alabel or indicating means capable of signaling the formation of animmunocomplex containing a polypeptide or antibody molecule of thepresent invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-diethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent oneclass of gamma ray emission-producing radioactive element indicatinggroups. Particularly preferred is ¹²⁵I. Another group of useful labelingmeans are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N which themselvesemit positrons. The positrons so emitted produce gamma rays uponencounters with electrons present in the animal's body. Also useful is abeta emitter, such as ¹¹¹In or ³H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect the quantity of hypocretin in a sample. “ELISA” refersto an enzyme-linked immunosorbent assay that employs an antibody orantigen bound to a solid phase and an enzyme-antigen or enzyme-antibodyconjugate to detect and quantify the amount of an antigen present in asample. A description of the ELISA technique is found in Chapter 22 ofthe 4th Edition of Basic and Clinical Immunology by D. P. Sites et al.,published by Lange Medical Publications of Los Altos, Calif. in 1982 andin U.S. Pat. No. 3,654,090; U.S. Pat. No. 3,850,752; and U.S. Pat. No.4,016,043, which are all incorporated herein by reference.

In some embodiments, a hypocretin polypeptide, an antibody or amonoclonal antibody of the present invention can be affixed to a solidmatrix to form a solid support that comprises a package in the subjectdiagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from anaqueous medium although other modes of affixation applicable to proteinsand polypeptides can be used that are well known to those skilled in theart. Exemplary adsorption methods are described herein.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.);agarose; beads of polystyrene beads about 1 micron (μ) to about 5millimeters (mm) in diameter available from Abbott Laboratories of NorthChicago, Ill.; polyvinyl chloride, polystyrene, cross-linkedpolyacrylamide, nitrocellulose- or nylon-based webs such as sheets,strips or paddles; or tubes, plates or the wells of a microliter platesuch as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microliter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems.

G. Cell Lines Expressing Hypocretin

The invention also includes a host cell transformed with a recombinantDNA (recombinant DNA) molecule of the present invention. The host cellcan be either procaryotic or eucaryotic, although eucaryotic cells arepreferred, particularly mammalian cells. Preferred cells are isolated,that is, substantially homogeneous and therefore free from other celltypes or other cells having a hypocretin protein expressed therein.

A cell expressing a hypocretin of this invention has a variety of usesaccording to this invention. Particularly preferred are uses for bulkproduction of hypocretin, for the purpose of providing immunogen forproduction of antibody, for supply of therapeutic protein, for directbinding or for screening pharmaceutical compound banks for the presenceof hypocretin receptor-specific ligands, i.e., in drug screening assaysas described herein. Thus, particularly preferred are cells containing arecombinant DNA molecule that expresses a hypocretin protein of thisinvention.

In one embodiment, a cell is produced for transplantation into a bodytissue, thereby expressing hypocretin and providing replacement therapy.The cell can be syngeneic, and typically will be a brain tissue-derivedcell, such as a hippocampal cell, neonatal brain tissue cell, glioma andthe like neuronal tissue cell. Transplantation is accomplished usingsurgical procedures available to a neurosurgeon where thetransplantation is to be made into the brain, brain stem or otherneurological tissues. In preferred embodiments, the cell contains avector for expressing the hypocretin in which the expression means isunder the control of a regulatable promoter, as is well known, such thatexpression of the hypocretin protein can be regulated.

Eucaryotic cells useful for expression of a hypocretin protein are notlimited, so long as the cell or cell line is compatible with cellculture methods and compatible with the propagation of the expressionvector and expression of the hypocretin protein gene product. Preferredeucaryotic host cells include yeast and mammalian cells, preferablyvertebrate cells such as those from a mouse, rat, monkey or humanfibroblastic cell line. Preferred eucaryotic host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 (ATCC CRL 1658), HELA cells (ATCC CCL 2),baby hamster kidney cells (BHK), COS-7, COS-1, HEK293 (ATCC CRL 1573),Ltk-1, AV-12 (ATCC CRL 9595), and the like eucaryotic tissue culturecell lines.

Transformation of appropriate cell hosts with a recombinant DNA moleculeof the present invention is accomplished by well known methods thattypically depend on the type of vector used. With regard totransformation of procaryotic host cells, see, for example, Cohen etal., Proc. Natl. Acad. Sci. USA, 69:2110 (1972); and Maniatis et al.,Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

With regard to transformation of vertebrate cells with vectorscontaining recombinant DNAs, see, for example, Graham et al., Virol.,52:456 (1973); Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76(1979), and the teachings herein.

Successfully transformed cells, i.e., cells that contain a recombinantDNA molecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrecombinant DNA of the present invention can be cloned to clonallyhomogeneous cell populations that contain the recombinant DNA. Cellsfrom those colonies can be harvested, lysed and their DNA contentexamined for the presence of the recombinant DNA using a method such asthat described by Southern, J. Mol. Biol., 98:503 (1975) or Berent etal., Biotech., 3:208 (1985).

In addition to directly assaying for the presence of recombinant DNA,successful transformation can be confirmed by well known immunologicalmethods when the recombinant DNA is capable of directing the expressionof hypocretin or by the detection of hypocretin binding activity.

For example, cells successfully transformed with an expression vectorproduce proteins displaying hypocretin antigenicity or biologicalactivity. Samples of cells suspected of being transformed are harvestedand assayed for either hypocretin biological activity or antigenicity.

In addition to the transformed host cells themselves, the presentinvention also includes a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Preferably, the culture alsocontains a protein displaying hypocretin antigenicity or biologicalactivity.

Nutrient media useful for culturing transformed host cells are wellknown in the art and can be obtained from several commercial sources. Inembodiments wherein the host cell is mammalian, a “serum-free” mediumcan be used.

H. Screening Methods to Identify Agonists and Antagonists of Hypocretin

The ability to selectively bind/modulate function of a hypocretinreceptor by a hypocretin ligand is at the heart of useful hypocretinpharmacology, and depends on identifying pharmacological molecules whichcan act a selective ligands, agonists or antagonists for a hypocretinreceptor. To that end, the elucidation of new hypocretin proteins, suchas those described herein, provides valuable tools for the search forselective reagents, tools that are useful in binding assays, and inscreening assays which indicate selective drug response to thehypocretin receptor.

The invention includes methods for determining whether a molecule bindsto, and preferably whether the molecule activates, a preselectedhypocretin receptor.

The method comprises conducting a binding assay to identify moleculeswhich bind the hypocretin receptor, as described in any of the assaysherein. Thus, the method comprises (1) contacting a candidate moleculewith a cell having a hypocretin receptor under conditions permittingbinding of hypocretin to the receptor, and (2) detecting the presence ofthe candidate molecule bound to the hypocretin receptor, therebydetermining whether the candidate binds to the receptor. The receptor istypically a cell surface protein when expressed by the cells.

Alternatively, one can use a competition format to identify analogs ofhypocretin by using a labeled hypocretin, and measuring the amount ofbound label in the presence of a candidate ligand, indicating whetherthe candidate competes with labeled hypocretin for binding to thereceptor. An exemplary competition assay is described herein.

It is also possible to use the above method to determine whether themolecule which binds to the hypocretin receptor also activates ormotivates the receptor's function, i.e., acts as an agonist, ordetermine whether the molecule inhibits the receptor's function, i.e.,acts as an antagonist, or acts as and inverse agonist. Thus, byevaluating in the detecting step whether the hypocretin receptor isactivated, one determines whether the candidate molecule is bioactive.

Methods for detecting bioactivity of the candidate molecule can vary,but typically involve measuring changes in intracellular levels of asecondary messenger effected as a result of binding, detecting changesin electrical potential, observing physiological or behavioral effectsrelated to hypocretin function, and the like methods. Exemplary assaysfor binding or for hypocretin-specific bioactivity are described in theExamples and include measurement of electrical changes of hypothalamicneurons, measurement of food intake or body temperature, or directbinding to a cell having a hypocretin receptor.

It is noted that the hypocretin receptor has not been characterized inextensive detail. Thus, any receptor that binds hypocretin can bereferred to as a hypocretin receptor for the purposes of a screeningassay, although receptors with the highest affinity and specificity forhypocretin are preferred. In practicing the present screening methods,one can use any of a variety of cells lines or tissues that possess ahypocretin receptor, including the exemplary cell lines and tissuesdescribed herein. The invention should not be construed as limiting solong as the binding or bioactivity assay involves the use of ahypocretin receptor. In preferred embodiments, a receptor that isspecific for hypocretin should be used. Specificity can be demonstratedby well known methods of ligand binding and ligand-mediated activation.

A related embodiment includes a method for screening to identify acandidate molecule that can bind, inhibit or activate a preselectedhypocretin receptor by functioning as a hypocretin agonist orantagonist. The method comprises:

(a) contacting a mammalian cell with said candidate drug underconditions permitting activation of said hypocretin receptor byhypocretin; and

(b) detecting the activation status of said hypocretin receptor, andthereby determining whether the drug activates or inhibits saidreceptor.

I. Methods for Altering Hypocretin Receptor Function

a. Therapeutic Methods

The certain reagents described in the present invention have thecapacity to modulate hypocretin receptor function, such as agonists orantagonists, and therefore are useful in therapeutic methods forconditions mediated by the hypocretin receptor.

Hypocretin polypeptides that mimic exposed regions of hypocretin havethe ability to function as analogs and compete for binding to thehypocretin receptor, or for other agents that would normally interactwith the receptor, thereby inhibiting binding of hypocretin to thereceptor.

Furthermore, antibodies and monoclonal antibodies of the presentinvention that bind to exposed regions of hypocretin have the capacityto alter hypocretin receptor function by blocking natural interactionswith hypocretin that normally interact at the site. Exemplary antibodiesare the anti-hypocretin antibodies described earlier.

Finally, oligonucleotides are described herein which are complementaryto mRNA that encodes a hypocretin protein of this invention and that areuseful for reducing gene expression and translation of the hypocretinmRNA, thereby altering hypocretin levels in a tissue.

In one embodiment, the present invention provides a method formodulating hypocretin function in an animal or human patient comprisingadministering to the patient a therapeutically effective amount of aphysiologically tolerable composition containing a hypocretinpolypeptide, analog or peptidomimetic, anti-hypocretin antibody ormonoclonal antibody, hypocretin agonist or antagonist, or anoligonucleotide of the present invention.

A therapeutically effective amount of a hypocretin polypeptide, as anexample for practicing the invention, is a predetermined amountcalculated to achieve the desired effect, i.e., to modulate receptorinteraction with its normal target, and thereby interfere with normalreceptor function. Depending on the structure of the particular peptidethe binding of some peptides will activate the receptor, while bindingof other peptides will not activate the receptor.

Similarly, a therapeutically effective amount of an anti-hypocretinantibody is a predetermined amount calculated to achieve the desiredeffect, i.e., to immunoreact with the hypocretin, and thereby inhibitthe hypocretin receptor's ability to interact with its normal target,hypocretin, and thereby interfere with normal receptor function.

The in vivo inhibition of hypocretin receptor function using ahypocretin polypeptide, an anti-hypocretin antibody, or hypocretinagonist or antagonist of this invention is a particularly preferredembodiment and is desirable in a variety of clinical settings, such aswhere the patient is exhibiting symptoms of an over or under activatedhypocretin receptor.

A therapeutically effective amount of a hypocretin polypeptide, agonistor antagonist of this invention is typically an amount such that whenadministered in a physiologically tolerable composition is sufficient toachieve a plasma concentration of from about 0.1 nanomolar (nM) to about100 nM, and preferably from about 0.5 nM to about 10 nM.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (μg) per milliliter (ml) toabout 100 μ/g/ml, preferably from about 1 μg/ml to about 5 μg/ml, andusually about 5 μg/ml.

The effectiveness of the therapy can be determined by observing ablationof the symptoms associated with the function of the hypocretin receptorbeing inhibited.

The therapeutic compositions containing a hypocretin polypeptide,agonist, antagonist or anti-hypocretin antibody of this invention areconventionally administered intravenously or by a method for delivery toa brain tissue, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

Delivery to a brain tissue or CSF can be accomplished by a variety ofmeans, including by direct injection, by use of a cannula into thetarget tissue, by direct application in a surgical procedure, byadsorption across the blood-brain barrier following intravenousadministration, by viral vectors, and the like means.

The therapeutic compounds and compositions are generally administered soas to contact the cells or the tissue containing cells which contain thetarget hypocretin receptor. This administration can be accomplished byintroduction of the composition internally such as orally,intravenously, intramuscularly, intranasally or via inhalation ofaerosols containing the composition, and the like, by cannula into abrain tissue, or by introduction into or onto a tissue system as byintroduction transdermally, topically or intralesionally, insuppositories, or by intra-orbital injection, and the like.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are particular to each individual. However, suitable dosage rangesfor systemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain concentrations in the CSF or blood inthe ranges specified for in vivo therapies are included.

As an aid to the administration of effective therapeutic amounts of ahypocretin polypeptide, agonist, antagonist, antibody, or monoclonalantibody, (hereinafter a “therapeutic agent”) a diagnostic method ofthis invention for detecting a therapeutic agent in the subject's CSF orblood is useful to characterize the fate of the administered therapeuticagent. Suitable diagnostic (monitoring) assays are described herein.

b. Methods for Inhibiting Gene Expression

In another embodiment, the invention includes the use of nucleic acidsencoding portions of a hypocretin gene for inhibiting gene expressionand function.

The present invention provides for a method for inhibiting expression ofhypocretin gene products and thereby inhibiting the function of thetarget hypocretin protein. The DNA segments and their compositions havea number of uses, and may be used in vitro or in vivo. In vitro, thecompositions may be used to block function and expression of hypocretinin cell cultures, tissues, organs and the like materials that canexpress hypocretin. In vivo, the compositions may be usedprophylactically or therapeutically for inhibiting expression of ahypocretin gene, and by inhibiting diseases or medical conditionsassociated with the expression or function of the hypocretin gene or theactivity state of its receptor.

The method comprises, in one embodiment, contacting cells or tissueswith a therapeutically effective amount of a pharmaceutically acceptablecomposition comprising a DNA segment of this invention. In a relatedembodiment, the contacting involves introducing the DNA segmentcomposition into cells expressing a hypocretin protein.

The DNA segment can be in a variety of forms, but is preferably in asingle-stranded form to facilitate complementary hybridization to thetarget mRNA in the cell in which the hypocretin gene expression is to bealtered.

The term “cells” is intended to include a plurality of cells as well assingle cells. The cells can be isolated, or can be cells that form alarger organization of cells to form a tissue or organ.

Another embodiment is a method of inhibiting the expression ofhypocretin genes in a patient comprising administration to the patientof a therapeutically effective amount of a DNA segment composition ofthis invention in a pharmaceutically acceptable excipients. In caseswhere the distribution of the hypocretin is believed to be disseminatedin the body, the administration of therapeutic oligonucleotide can besystemic. Alternatively, the target hypocretin can be localized to atissue, and the therapeutic method can likewise be directed atdelivering the therapeutic DNA segment to the tissue to be treated.

The concentration of the active DNA segment ingredient in a therapeuticcomposition will vary, depending upon the desired dosage, use, frequencyof administration, and the like. The amount used will be atherapeutically effective amount and will depend upon a number offactors, including the route of administration, the formulation of thecomposition, the number and frequency of treatments and the activity ofthe formulation employed.

The use of therapeutic DNA segments, and therefore the delivery of thoseDNA segments into cells where they are effective, has been described ina variety of settings. It is generally known that therapeuticallyeffective intracellular levels of nucleic acids, and particularlysmaller nucleic acids such as DNA segments and oligonucleotides, can beachieved by either exposing cells to solutions containing nucleic acidsor by introduction of the nucleic acids into the inside of the cell.Upon exposure, nucleic acids are taken up by the cell where they exerttheir effectiveness. In addition, direct introduction into the cell canbe provided by a variety of means, including microinjection, delivery bythe use of specific uptake vehicles, and the like.

The pharmaceutical composition containing the therapeuticoligonucleotide preferably also contains physiologically acceptablecarriers, in particular hydrophobic carriers which facilitate carryingthe oligonucleotide through the cell membrane or blood brain barrier.

Exemplary descriptions of the delivery of therapeutic DNA segments andoligonucleotides into cells can be found in the teachings of U.S. Pat.Nos. 5,04,820, 4,806,463, 4,757,055, and 4,689,320, which teachings arehereby incorporated by reference.

A therapeutically effective amount is a predetermined amount calculatedto achieve the desired effect, i.e., to bind to a hypocretin genepresent and thereby inhibit function of the gene.

As is apparent to one skilled in the art, the copy number of ahypocretin gene may vary, thereby presenting a variable amount of targetwith which to hybridize. Thus it is preferred that the therapeuticmethod achieve an intracellular concentration of a therapeutic DNAsegment of this invention in molar excess to the copy number of the genein the cell, and preferably at least a ten-fold, more preferably atleast a one-hundred fold, and still more preferably at least a onethousand-fold excess of therapeutic DNA segments relative to the genecopy number per cell. A preferred effective amount is an intracellularconcentration of from about 1 nanomolar (nM) to about 100 micromolar(μM), particularly about 50 nM to about 1 μM.

Alternatively, a therapeutically effective amount can be expressed as anextracellular concentration. Thus it is preferred to expose a cellcontaining a hypocretin gene to a concentration of from about 100 nM toabout 10 millimolar (mM), and preferably about 10 μM to 1 mM. Thus, inembodiments where delivery of a therapeutic DNA segment composition isdesigned to expose cells to the nucleic acid for cellular uptake, it ispreferred that the local concentration of the DNA segment in the area ofthe tissue to be treated reach the extracellular concentrations recitedabove.

For patient dosages, using a 20 nucleotide base double-stranded DNAsegment as the standard, a typical dosage of therapeutic composition fora 70 kilogram (kg) human contains in the range of about 0.1 milligram(mg) to about 1 gram of 20-mer DNA segment per day, and more usually inthe range of about 1 mg to 100 mg per day. Stated differently, thedosage is about 1 μg/kg/g day to about 15 mg/kg/day, and preferablyabout 15 to 1500 μg/kg/day.

The in vivo inhibition of hypocretin gene expression and function by atherapeutic composition of this invention is desirable in a variety ofclinical settings, such as where the patient is at risk for diseasebased on expression of the hypocretin gene.

c. Therapeutic Compositions

The present invention includes therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with a therapeutic reagent of this invention,namely a hypocretin polypeptide, an anti-hypocretin antibody ormonoclonal antibody, or oligonucleotide as described herein, dissolvedor dispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to amammal or human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions, however, solid forms suitable forsolution, or suspensions, in liquid prior to use can also be prepared.The preparation can also be emulsified.

The active ingredient can be mixed with excipient which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipient are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

As described herein, for intracellular delivery of oligonucleotides,specialized carriers may be used which facilitate transport of theoligonucleotide across the cell membrane. These typically arehydrophobic compositions, or include additional reagents which targetdelivery to and into cells.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an amount of a hypocretin polypeptideor anti-hypocretin antibody molecule of the present invention sufficientto inhibit hypocretin function. Typically this is an amount of at least0.1 weight percent, and more preferably is at least 1 weight percent, ofpeptide or antibody per weight of total therapeutic composition. Aweight percent is a ratio by weight of peptide or antibody to totalcomposition. Thus, for example, 0.1 weight percent is 0.1 grams ofpolypeptide per 100 grams of total composition.

The following Examples are illustrative of one means of practicingcertain aspects of the invention disclosed herein and should not beconstrued so as impart any undue limitations upon the invention asclaimed below.

EXAMPLE 1

Young adult Sprague-Dawley rats of both genders were sacrificed underanesthesia by decapitation and their brains quickly removed. Thehypothalamus, hippocampus, and cerebellum were immediately dissected onan ice-cold plate following the boundaries described by Glowinski andIversen (Glowinski, J., & Iversen, L. L., J. Neurochem. 13:655-669,1966). The block of hypothalamic tissue was 2 mm deep and was takenusing the optic chiasm as the rostral limit and the mammillary bodies ascaudal reference. Cytoplasmic RNA was isolated rapidly from thedissected tissues (Schibler, K., Tosi, M., Pittet, A. C., Fabiani, L., &Wellauer, P. K., J. Mol. Biol. 142:93-116, 1980) and enriched forpoly(A)-containing species by oligo(dT)-cellulose chromatography (Aviv,H., & Leder, P., Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). For theNorthern blots, RNA was isolated (Chirgwin, J. M., Przybyla, A. E.,MacDonald, R. J., & Rutter, W. J., Biochemistry 18:5294, 1979) fromfrozen tissue purchased from Zivic-Miller (Zelienople, Pa.). cDNAlibraries were prepared as described previously Usui et al, supra,except that pBCSK⁺ was used for the subtracted library rather thanpT7T3D because lower backgrounds have been found in the subsequent stepsusing the former vector (H. Usui, personal communication). The number ofrecombinants in the libraries were: pT7T3D hypothalamus 8×10⁶;cerebellum pGEM11Zf (−) 5×10⁵; hippocampus pGEM11Zf(−) 1×10⁶.

Subtractive hybridization was performed in two cycles using thepreviously described procedure (Usui et al., supra). Briefly, 1 μg oftrace-labeled, tagged hypothalamus target cDNA prepared as describedfrom the pT7T3D target library was annealed for 24 hrs at 68 degreeCelsius in 10 μl of hybridization buffer (Usui et al, supra) with 20 μgcerebellum cRNA (ratio 1:20). After hydroxyapatite chromatography, thesingle-stranded fraction corresponded to 10% of the input material, asjudged by tracer quantitation. This was mixed with 20 μg of hippocampuscRNA (estimated ratio 1:200) for a second 24 hr hybridization, afterwhich 30% of the input chromatographed at the single-strand position.Cumulatively, these steps removed more than 97% of the input tracer. Analiquot of the single-stranded material was used as template in a30-cycle PCR (program: 94 degree Celsius for 15 sec, 60 degree Celsiusfor 15 sec, 72 degree Celsius for 1 min) using primers corresponding tothe tag sequences (Usui et al., supra): 5′-AACTGGAAGAATTCGCGG-3′ (SEQ IDNO: 19) and 5′-AGGCCAAGAATTCGGCACGA-3′ (SEQ ID NO: 16). Theamplification product was cleaved with NotI, then EcoRI, and insertedinto pBCSK⁺. A dot blot was prepared and screened with probes preparedfrom the target, subtracted target and driver libraries as previouslydescribed by Usui et al, supra, using serial dilutions of plasmid cDNAclones isolated previously in this laboratory. The target and subtractedtarget cDNA libraries were screened to determine the frequency ofoxytocin and VAT-1 cDNA clones using as probes clones isolated in thepresent study.

Clone 35 cDNA from the subtracted rat hypothalamus library was used as aprobe to screen a rat brain cDNA library in the plasmid pHG327 asdescribed by Forss-Petter et al., J. Mol. Neurosci. 1:63-75 (1989). ThecDNA library was constructed as described by Staeheli et al., Cell44:147-158 (1986).

EXAMPLE 2

Similarly, following the procedures of Example 1, a mouse (C57/B16)hypothalamus cDNA library, constructed in the pT7T3D vector, was used asa template for PCR amplification (primers 5′ TAAGACGACGGCCTCAG 3′, SEQID NO: 17, and 5′ CACACCAACAGAGAAACG 3′, SEQ ID NO: 18) to obtain themouse homolog of the rat H35 cDNA obtained above. The mouse and rat cDNA(SEQ ID NO: 15 and SEQ ID NO: 14, respectively) and protein sequences(mouse SEQ ID NO:2; rat SEQ ID NO: 1) are compared in FIG. 5. The 569nucleotide rat sequence has the potential to encode a 130-residueputative secretory protein (preprohypocretin) with an apparent signalsequence and 3 additional sites for potential proteolytic maturation(FIG. 5A). Two of the putative products of proteolysis (hcrt1, SEQ IDNO: 7 and hcrt2, SEQ ID NO: 9) have 14 amino acid identities across 20residues (FIG. 5B). This region of one of the peptides contains a 7/7match with secretin (FIG. 5B, SEQ ID NO: 21), suggesting that theprepropeptide gives rise to two peptide products that are structurallyrelated both to each other and to secretin.

The mouse hypocretin nucleotide sequence (SEQ ID NO: 15) differs in 35positions relative to the rat (SEQ ID NO: 14), and contains 16additional nucleotides near its 3′ end. Of these differences, 19 arewithin the putative protein-coding region (FIG. 5A), only 7 of whichaffect the encoded protein sequence: one amino acid difference atresidue 3 is a neutral substitution in the apparent secretion signalsequence; the remaining 6 differences are near the C-terminus, one ofwhich obliterates a potential proteolytic cleavage site. The absence ofthis site and the nature of the other differences make it unlikely thattwo of the four possible rat maturation products are generated andfunctional in mice. However, the two putative hcrt peptides that arerelated both to each other and to the secretin family are absolutelypreserved between the two species, providing strong support for thenotion that these peptides have a function conserved during evolution.Both hcrt1 and hcrt2 terminate with glycine residues, leaving thenitrogen of the terminal glycine as a C-terminal amide in the maturepeptide.

Several hypocretin peptides are distinguished within the sequence ofhypocretin (FIG. 5). The peptide from about amino acid residue 28 toabout amino acid residue 130 (SEQ ID NO:6) represents the peptideproduced by cleavage of the signal peptide. The peptide from about aminoacid residue 28 to about amino acid residue 66 (SEQ ID NO:7) correspondsto hcrt1. The peptide from about amino acid residue 28 to about aminoacid residue 65 (SEQ ID NO:8) corresponds to hcrt1 matured bypeptidylglycine alpha-amidating monooxygenase, leaving the nitrogen ofthe terminal glycine as a C-terminal amide in the mature peptide. Thepeptide from about amino acid residue 70 to about amino acid residue 97(SEQ ID NO:9) corresponds to hcrt2. The peptide from about amino acidresidue 70 to about amino acid residue 96 (SEQ ID NO:10) corresponds tohcrt2 matured by peptidylglycine alpha-amidating monooxygenase, leavingthe nitrogen of the terminal glycine as a C-terminal amide in the maturepeptide. The peptide from about amino acid residue 47 to about aminoacid residue 66 (SEQ ID NO:11) corresponds to the consensus sequenceregion of hcrt1 (FIG. 5B). The peptide from about amino acid residue 78to about amino acid residue 97 (SEQ ID NO:12) corresponds to theconsensus region of Hrct2. The peptide GNHAAGILT (SEQ ID NO:13) iscommon to both hcrt1 and hcrt2.

EXAMPLE 3

Rat H35 (SEQ ID NO:3) is inserted into the BamH1 sites of a pHG237vector. Upon digestion with BamH1 restriction enzyme, the resultant 569bp fragment is then inserted directly into the BglII site of thepolylinker region of the pCM 4 vector (D. Russell, U. Texas SouthwesternMedical Center, Dallas, Tex.), which uses the cytomegalovirus (CMV)promoter. Several eight to ten amino acid epitope tags are added by PCRto the C-terminus of H35 to allow visualization of the expressedproduct.

The respective 5′ and 3′ primers, 5′ ATCGAGATCTAGACACCATGAACCTTCCTTCTACAAAGGTT 3′ (SEQ ID NO: 22) and 5′ ACTGTCTAGATCATAGATCTTCTTCAGAAATAAGTTTTTGTTCGACTCTGGATCCGCCCCGGG GCGCT 3′ (SEQ IDNO: 23), are used as primers to amplify H35 beginning at position 85 inSEQ ID NO:3 with an inserted BglII site added at its 5′ end to the 3′end having an inserted c-myc epitope tag. The PCR products are subclonedinto pCMV and transfected into a mammalian host cell to produce anH35-myc tagged protein product.

H35 proteins are also produced in bacteria by subcloning the H35 codingsequence into pRSET B (Invitrogen, San Diego, Calif.), which encodes sixhistidines prior to the H35 sequence. The vector contains a T7 promoterwhich drives expression of 6×His-tagged proteins in E. coli. Therespective 5′ and 3′ oligonucleotides 5′ ATCGAGATCTCTTGGGGTGGACGCGCAGCCT3′ (SEQ ID NO: 24) and 5′ ACTGAATTCTCAGACTCTGGATCCGCCCCG 3′ (SEQ ID NO:25) are used as PCR primers to amplify the rat H35 sequence into theBglII and EcoRI sites of the pRSET B vector. The resultinghypocretin-poly-(His) fusion protein may be purified by affinitychromatography on a metal affinity resin.

An H35-glutathione-S-transferase fusion protein is produced in E. coliby subcloning the H35 sequence into a pGEX2 vector (Pharmacia).

EXAMPLE 4

The mouse Hcrt gene was mapped to Chromosome 11 using an interspecificbackcross. A single-strand sequence polymorphism between C57BL/6J andSPRET/Ei was detected as previously described and mapped on The JacksonLaboratory BSS panel. An Hcrt-specific product of approximately 600 basepairs was amplified from mouse C57BL/6J genomic DNA using syntheticoligonucleotides 5′-GACGGCCTCAGACTTCTTGG-3′ (SEQ ID NO: 26) and5′-GCAACAGTTCGTAGAGACGG-3′ (SEQ ID NO: 27). This product contained aputative intron, and its identity as hcrt was confirmed by sequencing(data not shown). Genotype data and references for these and otherlinked markers can be accessed via the Mouse Genome Database(http://www.informatics.jax.org).

No recombinants in 94 BSS mice were found between hcrt and thepreviously mapped loci Brca1, Tubg and Mpmv8, placing Hcrt maximallywithin 3.8 cM (95% confidence limit) of these genes. The Hoxb cluster isapproximately 1 cM centromeric to Hcrt, and the Kcnj2 gene is locatedapproximately 4 cM telomeric. Hcrt is located in the portion of mouseChromosome 11 that shows conserved synteny with human Chromosome17q21-q24.

In Northern blot studies using poly(A)⁺ RNA prepared from brain anddifferent peripheral tissues, the 700-nucleotide hypocretin mRNA wasdetected only in brain samples. Previous studies with RNA from differentregions of the brain had detected the hypocretin mRNA predominantly inhypothalamus samples. In samples of RNA from whole brains of developingrats, hypocretin mRNA was detected at low concentrations as early asembryonic day 18, but increased in concentration dramatically after thethird postnatal week. There was no detectable difference between brainsamples from adult males and females, suggesting that the late onset wasnot related to sexually dimorphic processes. In situ hybridizationstudies detected cell bodies in the dorsal-lateral hypothalamus and incells that line the ventricles.

EXAMPLE 5

A polyclonal antiserum (serum 2050) was raised to a chemicallysynthesized peptide corresponding to the C-terminal 17 amino acidresidues (CPTATATACAPRGGSRV, SEQ ID NO: 28) of the rat preprohypocretinsequence. In Western transfer blots using as target electrophoreticallyseparated proteins from bacteria transformed with the plasmid pRSET Bengineered to express preprohypocretin, a single prominentimmunoreactive band was observed with a migration of approximately 19kDa with the hyperimmune serum, but not with the preimmune serum. Noimmunoreaction was detected with an extract from bacteria transformedwith a preprohypocretin/pRSET B expression plasmid, indicating thatdetection of the 19 kDa target requires hypocretin expression. Analogousresults were obtained with an additional antiserum to the 17 mer and twoantisera to synthetic hcrt2.

In immunohistochemical studies with antiserum 2050 on sections fromperfused adult male rats, immunoreactive cell bodies were observedexclusively in the perifornical nucleus and dorsal and lateralhypothalamic areas, consistent with the in situ hybridization results(FIG. 4). This coincident staining, its elimination when the serum waspreincubated with the peptide immunogen, and the very low nonspecificbackground observed, together with the Western blot results, providedstrong evidence for the specificity of the antiserum for hypocretin. Inaddition to cell bodies, the serum detected a prominent network offibers located within the hypothalamus, particularly the posteriorregion. Less prominent fiber projections were observed in apparentterminal fields within the preoptic area, the medial dorsal and reuniensnuclei of the thalamus, the dorsal raphe nucleus, the locus coeruleus,the laterodorsal tegmental nucleus, the central gray, the colliculi andthe nucleus of the solitary tract. In immuno-electron microscopystudies, immunoreactive secretory vesicles were observed.

EXAMPLE 6

The putative structures of the hypocretins, their expression within thedorsolateral hypothalamus and accumulation within fibers and vesiclessuggested that they may have intercellular signaling activity. To testthis hypothesis, 10-day cultures of synaptically-coupled rathypothalamic neurons were prepared and postsynaptic currents wererecorded under voltage clamp. Application of a synthetic peptidecorresponding to amidated hcrt2 at 1 μM evoked a substantial, butreversible, increase in the frequency of postsynaptic currents in 75% ofthe neurons tested (FIG. 7A), indicating an increase in the activity ofpresynaptic axons, and suggesting an increase in excitation. The other25% of the cells showed no response to hcrt2. There was little responseby hypothalamic neurons that had been in culture for only 3-5 days,suggesting that a certain degree of synaptic maturity was required forthe effect. Hcrt2 elicited no response from synaptically coupledhippocampal dentate granule neurons in culture, demonstrating targetselectivity and suggesting that specific receptors for hcrt2 may exist.

EXAMPLE 7

Synthetic, amidated hcrt2 peptide at different concentrations wasinfused intracerebroventricularly in rats and body temperature by wasmonitored telemetry. Stereotactic ablation studies have previouslyimplicated the dorsal-lateral hypothalamus in feeding behavior, bloodpressure, and central regulation of immune function, although precisenuclei have not been correlated with these activities. A threshold-typeresponse was obtained in which, at the highest dose, 10 μg, bodytemperature dropped from 37.7 to 36.7 degrees Celsius over 30 minutesfollowing administration, then recovered to normal over 2 hours. Foodintake was monitored over 2 hours following administration and a 40%reduction in food intake was measured at a dose of 5 μg. Whereas theconcentrations of peptide required for an effect might seem high, theyare comparable to the doses of leptin administered ICV to obtain acomparable suppression of food intake. The presumed target cells ofhypocretin may not be very accessible by this unphysiological mode ofadministration. Local injection or intravenous administration ofhypocretin might be more suitable for physiological studies.

The cell bodies that produce the hypocretins are located in an areaimplicated in ablation studies as regulatory centers for appetitivebehaviors, suggesting that the hypocretins may serve as a majortransmitters for the central system signalling the status of energybalance in the major fat repositories. The projections ofhypocretin-producing cells indicate that the peptides function bothwithin the hypothalamus and at a complex and diffuse network of targetsin several regions of the brain that may coordinate the various aspectsof appetitive behavior, adaptive thermogenesis and metabolic regulation.

Rat hypothalamus from 18-day embryos was cultured for 10 days in vitro.The mediobasal hypothalamus was removed from embryonic day 18 SpragueDawley rats. The tissue was enzymatically digested in a mild proteasesolution (10 U/ml papain and 0.2 mg/ml L-cysteine in Earle's balancedsalt solution) for 30 minutes. Next, the tissue was pelleted, and theprotease solution was removed. Tissue was then suspended in standardtissue culture medium (glutamate- and glutamate-free DMEM supplementedwith 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 6gm/l glucose) and then triturated into a single-cell suspension. Cellswere washed and pelleted an additional three times. The single-cellsuspension was plated onto 22 mm² glass coverslips that had been coatedwith high-molecular-weight (540,000 Da) poly-D-lysine. High-densitycultures (200,000/cm²) were used for all experiments. Hypothalamicneural cultures were maintained in a Napco 3600 incubator (37 degreeCelsius and 5% CO₂) until they were ready for use. To limit non-neuronalcell proliferation cytosine arabinofuranoside (1 μM) was added to thetissue culture medium 1 day after plating.

Synaptically coupled hypothalamic neurons were recorded in voltage clampwith a whole cell pipette (holding potential=−60 mV). This recording istypical of 9 of 12 cells examined under these conditions. The frequencyof postsynaptic events (PSCs) was greatly increased (up to +400%) by 1μM hcrt2 applied to the bath. After washout of the peptide, thefrequency of PSCs returned to normal baseline levels. Inset boxes showhigher resolution of the events indicated by the dotted line. Both boxes(in FIGS. 7A and B) were recorded with an identical delay after hcrt2administration. A less mature hypothalamic neuron after 4 days ofculture was unresponsive to 1 μM hcrt2 (FIG. 7B). Pipette solutioncontained 128 mM KMeSO₄, 27 mM KCl, 0.4 mM EGTA, 1 mM ATP, and 0.5 mMGTP.

The preceding written description provides a full, clear, concise andexact disclosure of the invention so as to enable one skilled in the artto make and use the same. This disclosure should not be construed so asto impart any direct or implied limitation upon the scope of theinvention which is particularly pointed out and distinctly claimedbelow.

TABLE 1 Cumulative Data From 100 Clones Clone^(a) BLAST Homology^(b)Accession#^(c) #^(d) Pattern^(e)  2 + oxytocin M25649 13 A/A  6 +VAT1-like T05306 11 B/B  1 + CART U10071 7 C 35 + novel 6 A/A 15 + novel4 B/C 25 + POMC J00759 4 A 12 + novel(E) R75926 3 B/B 16 + vasopressinM25646 3 A 18 + glutathione perox U13705 3 B 29 + novel CaM kinase 3 B/C 3 + novel 2 B/C 10 + novel 2 B/B 51 + ubiquitin carrier M91679 2 C 62 +novel 2 C  5 − calbindin U08290 B 14 + melanin-conc hormone M62641 C17 + asp aminotrans M18467 D 19 − novel(E) R74893 D 20 + novel A/B 21 −novel(E) T32756 A/D 22 − novel D 33 + novel(E) R67552 A/A 34 − Cl/HCO₃exchanger J05167 C 37 + novel B/D 39 − novel C 45 + novel C 46 +fibromodulin X82152 C 47 perox enolhydratase U08976 C 48 + galaninJ03624 B 52 − 5-HT₂ receptor L31546 B 53 + MHC orf M32010 E 55 + HNFdimer cofactor M83740 C 56 + carbonyl reductase X84349 C 57 + tyrosinehydroxylase M10244 A 63 + novel D 67 + novel B/B 73 + novel C 74 +novel(E) T93996 C 75 + lamin C2 D14850 A 86 + novel C 92 − novel(E)R49544 C 98 + novel B/D 99 − neuronal kinesin U06698 B/D ^(a) number ofprototype clone in set of 100 followed by indication (+/−) as to whether3′ sequence contained poly(A)-addition hexad (no 3 ′ sequence for clone47) ^(b) short name of matching species or novel for no match:(E)indicates EST match ^(c) GenBank database reference ^(d) number ofrepresentatives in set of 100 ^(e) hybridization pattern in cDNA librarySouthern assay/Northern blot assay. Code:A, target only; B, targethighly enriched; C, hypothalamus and hippocampus; D, not highlyenriched; E, too faint to categorize

29 1 130 PRT ratus ratus 1 Met Asn Leu Pro Ser Thr Lys Val Pro Trp AlaAla Val Thr Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Pro Pro Ala Leu LeuSer Leu Gly Val Asp Ala 20 25 30 Gln Pro Leu Pro Asp Cys Cys Arg Gln LysThr Cys Ser Cys Arg Leu 35 40 45 Tyr Glu Leu Leu His Gly Ala Gly Asn HisAla Ala Gly Ile Leu Thr 50 55 60 Leu Gly Lys Arg Arg Pro Gly Pro Pro GlyLeu Gln Gly Arg Leu Gln 65 70 75 80 Arg Leu Leu Gln Ala Asn Gly Asn HisAla Ala Gly Ile Leu Thr Met 85 90 95 Gly Arg Arg Ala Gly Ala Glu Leu GluPro Tyr Pro Cys Pro Gly Arg 100 105 110 Arg Cys Pro Thr Ala Thr Ala ThrAla Leu Ala Pro Arg Gly Gly Ser 115 120 125 Arg Val 130 2 130 PRT musmusculus 2 Met Asn Phe Pro Ser Thr Lys Val Pro Trp Ala Ala Val Thr LeuLeu 1 5 10 15 Leu Leu Leu Leu Leu Pro Pro Ala Leu Leu Ser Leu Gly ValAsp Ala 20 25 30 Gln Pro Leu Pro Asp Cys Cys Arg Gln Lys Thr Cys Ser CysArg Leu 35 40 45 Tyr Glu Leu Leu His Gly Ala Gly Asn His Ala Ala Gly IleLeu Thr 50 55 60 Leu Gly Lys Arg Arg Pro Gly Pro Pro Gly Leu Gln Gly ArgLeu Gln 65 70 75 80 Arg Leu Leu Gln Ala Asn Gly Asn His Ala Ala Gly IleLeu Thr Met 85 90 95 Gly Arg Arg Ala Gly Ala Glu Leu Glu Pro His Pro CysSer Gly Arg 100 105 110 Gly Cys Pro Thr Val Thr Thr Thr Ala Leu Ala ProArg Gly Gly Ser 115 120 125 Gly Val 130 3 569 DNA ratus ratus 3taagacgacg gcctcagact ccttgggtat ttggaccact gcaccgaaga taccatctct 60ccggattacc tctccctgag ctccagacac catgaacctt ccttctacaa aggttccctg 120ggccgccgtg acgctgctgc tgctgctact gctgccgccg gcgctgctgt cgcttggggt 180ggacgcgcag cctctgcccg actgctgtcg ccagaagacg tgttcctgcc ggctctacga 240actgttgcac ggagctggca accacgccgc gggcatcctc actctgggaa agcggcgacc 300tggaccccca ggcctccaag gacggctgca gcgcctcctt caggccaacg gtaaccacgc 360agctggcatc ctgaccatgg gccgccgcgc aggcgcagag ctagagccat atccctgccc 420tggtcgccgc tgtccgactg caaccgccac cgctttagcg ccccggggcg gatccagagt 480ctgaacccgt cttctatccc tgtcctagtc ctaactttcc cctctcctcg ccagtcccta 540ggcaataaag acgtttctct gttggtgtg 569 4 582 DNA mus musculus 4 taagacgacggcctcagact tcttgggtat ttggaccact gcactgaaga gatcatctct 60 ccagattactttcccctgag ctccaggcac catgaacttt ccttctacaa aggttccctg 120 ggccgccgtgacgctgctgc tgctgctact gctgccaccg gcgctgctgt cgcttggggt 180 ggacgcacagcctctgcccg actgctgtcg ccagaagacg tgttcctgcc gtctctacga 240 actgttgcacggagctggca accacgctgc gggtatcctg actctgggaa agcggcggcc 300 tggacctccaggcctccagg gacggctgca gcgcctcctt caggccaacg gtaaccacgc 360 agctggcatcctgaccatgg gccgccgcgc aggcgcagag ctagagccac atccctgctc 420 tggtcgcggctgtccgaccg taactatcac cgctttagca ccccggggag ggtccggagt 480 ttgaacccatcttctatcct tgtcctgatc caaacttccc cctctgctcg ccgctgtcag 540 tctcttggtaaatggcaata aagacgtttc tctgttggtg tg 582 5 1458 DNA ratus ratus 5gctaggagac attgcggcgg cggtggcggc gttggcagca gctgcagaca tgctgctgct 60caagaaacag acggaggaca tcagcagtgt ctatgagatc cgggagaagc tgggctcggg 120tgccttctct gaggtgatgc tggcccagga aaggggctct gctcatcttg tggccctcaa 180gtgcattccc aagaaagcac ttcggggcaa ggaggccctg gtggagaatg agatcgcagt 240actccgcagg attagccacc ccaacattgt ggctctggag gacgtccacg agagcccttc 300ccatctctac ttggccatgg agctggtaac aggtggtgaa ctgtttgacc gaatcatgga 360gcggggctcc tacacagaga aggatgcgag ccaccttgta gggcaggtcc ttggtgctgt 420ctcctacctt catagcctgg gcatcgtgca ccgggacctc aagcctgaaa acctcctcta 480tgccacacct tttgaggact ccaagatcat ggtctctgac tttggcctgt ccaaaattca 540agctggcaac atgctaggca cagcctgtgg gaccccagga tatgtggccc cagagctcct 600ggagcagaaa ccctacggga aggccgtaga tgtgtgggcc ctgggtgtca tctcctacat 660cctgctgtgt gggtaccccc ccttctatga tgagagcgat cctgaactct tcagccagat 720tctgagggcc agctacgagt ttgactctcc cttttgggat gacatctcag aatcagccaa 780agacttcatt cggcaccttc tggaacgtga tccccagaag aggttcacct gccaacaggc 840cttacagcat ctctggatct ctggggatgc agccttggac agggacatcc taggttctgt 900cagtgagcag atccagaaga attttgccag gacccactgg aagcgtgcat tcaatgccac 960atcattccta cgtcacatcc gtaagctggg acagagccca gagggtgagg aggcctccag 1020gcagggtatg acccgtcaca gccacccagg ccttgggact agccagtctc ccaagtggtg 1080acaaccaggt ggatgccaag gaaggccaag tggactgact cctagctttt ctttcctcca 1140gcccttttga tctccttccc tgatccttgt cccccggact ggcctctgtt ggaaagtcca 1200agaccgtggg tgtgatgcat ggcactgggg tatggggctt cccaagtatg tccccagcct 1260ctgtcctttg ttgctgccac cctctatgga aactgaggag gtattcaaaa atggatttgg 1320gggccatcct tcctgcacct tgcacgcaca tatgcattgc gtggctgttc tgtgctttgc 1380tgactgtggg tggtcctgct tgtgttgtag ccctttagtt cctcctcttt ccaaccaata 1440aagacaaaca gaacaatg 1458 6 103 PRT ratus ratus 6 Leu Gly Val Asp Ala GlnPro Leu Pro Asp Cys Cys Arg Gln Lys Thr 1 5 10 15 Cys Ser Cys Arg LeuTyr Glu Leu Leu His Gly Ala Gly Asn His Ala 20 25 30 Ala Gly Ile Leu ThrLeu Gly Lys Arg Arg Pro Gly Pro Pro Gly Leu 35 40 45 Gln Gly Arg Leu GlnArg Leu Leu Gln Ala Asn Gly Asn His Ala Ala 50 55 60 Gly Ile Leu Thr MetGly Arg Arg Ala Gly Ala Glu Leu Glu Pro Tyr 65 70 75 80 Pro Cys Pro GlyArg Arg Cys Pro Thr Ala Thr Ala Thr Ala Leu Ala 85 90 95 Pro Arg Gly GlySer Arg Val 100 7 39 PRT ratus ratus 7 Leu Gly Val Asp Ala Gln Pro LeuPro Asp Cys Cys Arg Gln Lys Thr 1 5 10 15 Cys Ser Cys Arg Leu Tyr GluLeu Leu His Gly Ala Gly Asn His Ala 20 25 30 Ala Gly Ile Leu Thr Leu Gly35 8 38 PRT ratus ratus 8 Leu Gly Val Asp Ala Gln Pro Leu Pro Asp CysCys Arg Gln Lys Thr 1 5 10 15 Cys Ser Cys Arg Leu Tyr Glu Leu Leu HisGly Ala Gly Asn His Ala 20 25 30 Ala Gly Ile Leu Thr Leu 35 9 28 PRTratus ratus 9 Pro Gly Pro Pro Gly Leu Gln Gly Arg Leu Gln Arg Leu LeuGln Ala 1 5 10 15 Asn Gly Asn His Ala Ala Gly Ile Leu Thr Met Gly 20 2510 27 PRT ratus ratus 10 Pro Gly Pro Pro Gly Leu Gln Gly Arg Leu Gln ArgLeu Leu Gln Ala 1 5 10 15 Asn Gly Asn His Ala Ala Gly Ile Leu Thr Met 2025 11 20 PRT ratus ratus 11 Arg Leu Tyr Glu Leu Leu His Gly Ala Gly AsnHis Ala Ala Gly Ile 1 5 10 15 Leu Thr Leu Gly 20 12 20 PRT ratus ratus12 Arg Leu Gln Arg Leu Leu Gln Ala Asn Gly Asn His Ala Ala Gly Ile 1 510 15 Leu Thr Met Gly 20 13 9 PRT ratus ratus 13 Gly Asn His Ala Ala GlyIle Leu Thr 1 5 14 393 DNA ratus ratus 14 atgaaccttc cttctacaaaggttccctgg gccgccgtga cgctgctgct gctgctactg 60 ctgccgccgg cgctgctgtcgcttggggtg gacgcgcagc ctctgcccga ctgctgtcgc 120 cagaagacgt gttcctgccgtctctacgaa ctgttgcacg gagctggcaa ccacgccgcg 180 ggcatcctca ctctgggaaagcggcgacct ggacccccag gcctccaagg acggctgcag 240 cgcctccttc aggccaacggtaaccacgca gctggcatcc tgaccatggg ccgccgcgca 300 ggcgcagagc tagagccatatccctgccct ggtcgccgct gtccgactgc aaccgccacc 360 gctttagcgc cccggggcggatccagagtc tga 393 15 393 DNA mus musculus tag sequence 15 atgaactttccttctacaaa ggttccctgg gccgccgtga cgctgctgct gctgctactg 60 ctgccaccggcgctgctgtc gcttggggtg gacgcacagc ctctgcccga ctgctgtcgc 120 cagaagacgtgttcctgccg tctctacgaa ctgttgcacg gagctggcaa ccacgctgcg 180 ggtatcctgactctgggaaa gcggcggcct ggacctccag gcctccaggg acggctgcag 240 cgcctccttcaggccaacgg taaccacgca gctggcatcc tgaccatggg ccgccgcgca 300 ggcgcagagctagagccaca tccctgctct ggtcgcggct gtccgaccgt aactatcacc 360 gctttagcaccccggggagg gtccggagtt tga 393 16 20 DNA Artificial Sequence tag sequence16 aggccaagaa ttcggcacga 20 17 17 DNA mus musculus 17 taagacgacg gcctcag17 18 18 DNA mus musculus 18 cacaccaaca gagaaacg 18 19 18 DNA ArtificialSequence tag sequence 19 aactggaaga attcgcgg 18 20 14 PRT mus musculus20 Arg Leu Leu Leu Gly Asn His Ala Ala Gly Ile Leu Thr Gly 1 5 10 21 36PRT ratus ratus 21 His Ser Asp Gly Thr Phe Thr Ser Lys Leu Ser Arg LeuArg Asp Ser 1 5 10 15 Ala Arg Leu Gln Arg Leu Leu Gln Gly Leu Val HisSer Asp Gly Thr 20 25 30 Phe Thr Ser Lys 35 22 41 DNA ratus ratus 22atcgagatct agacaccatg aaccttcctt ctacaaaggt t 41 23 68 DNA ratus ratus23 actgtctaga tcatagatct tcttcagaaa taagtttttg ttcgactctg gatccgcccc 60ggggcgct 68 24 31 DNA ratus ratus 24 atcgagatct cttggggtgg acgcgcagcc t31 25 30 DNA ratus ratus 25 actgaattct cagactctgg atccgccccg 30 26 20DNA mus musculus 26 gacggcctca gacttcttgg 20 27 20 DNA mus musculus 27gcaacagttc gtagagacgg 20 28 17 PRT ratus ratus 28 Cys Pro Thr Ala ThrAla Thr Ala Cys Ala Pro Arg Gly Gly Ser Arg 1 5 10 15 Val 29 358 PRTratus ratus 29 Met Leu Leu Leu Lys Lys Gln Thr Glu Asp Ile Ser Ser ValTyr Glu 1 5 10 15 Ile Arg Glu Lys Leu Gly Ser Gly Ala Phe Ser Glu ValMet Leu Ala 20 25 30 Gln Glu Arg Gly Ser Ala His Leu Val Ala Leu Lys CysIle Pro Lys 35 40 45 Lys Ala Leu Arg Gly Lys Glu Ala Leu Val Glu Asn GluIle Ala Val 50 55 60 Leu Arg Arg Ile Ser His Pro Asn Ile Val Ala Leu GluAsp Val His 65 70 75 80 Glu Ser Pro Ser His Leu Tyr Leu Ala Met Glu LeuVal Thr Gly Gly 85 90 95 Glu Leu Phe Asp Arg Ile Met Glu Arg Gly Ser TyrThr Glu Lys Asp 100 105 110 Ala Ser His Leu Val Gly Gln Val Leu Gly AlaVal Ser Tyr Leu His 115 120 125 Ser Leu Gly Ile Val His Arg Asp Leu LysPro Glu Asn Leu Leu Tyr 130 135 140 Ala Thr Pro Phe Glu Asp Ser Lys IleMet Val Ser Asp Phe Gly Leu 145 150 155 160 Ser Lys Ile Gln Ala Gly AsnMet Leu Gly Thr Ala Cys Gly Thr Pro 165 170 175 Gly Tyr Val Ala Pro GluLeu Leu Glu Gln Lys Pro Tyr Gly Lys Ala 180 185 190 Val Asp Val Trp AlaLeu Gly Val Ile Ser Tyr Ile Leu Leu Cys Gly 195 200 205 Tyr Pro Pro PheTyr Asp Glu Ser Asp Pro Glu Leu Phe Ser Gln Ile 210 215 220 Leu Arg AlaSer Tyr Glu Phe Asp Ser Pro Phe Trp Asp Asp Ile Ser 225 230 235 240 GluSer Ala Lys Asp Phe Ile Arg His Leu Leu Glu Arg Asp Pro Gln 245 250 255Lys Arg Phe Thr Cys Gln Gln Ala Leu Gln His Leu Trp Ile Ser Gly 260 265270 Asp Ala Ala Leu Asp Arg Asp Ile Leu Gly Ser Val Ser Glu Gln Ile 275280 285 Gln Lys Asn Phe Ala Arg Thr His Trp Lys Arg Ala Phe Asn Ala Thr290 295 300 Ser Phe Leu Arg His Ile Arg Lys Leu Gly Gln Ser Pro Glu GlyGlu 305 310 315 320 Glu Ala Ser Arg Gln Gly Met Thr Arg His Ser His ProGly Leu Gly 325 330 335 Thr Ser Gln Ser Pro Lys Trp Val Thr Thr Arg TrpMet Pro Arg Lys 340 345 350 Ala Lys Trp Thr Asp Ser 355

We claim:
 1. A polynucleotide comprising the sequence of SEQ ID NO:5. 2.A vector comprising the sequence of SEQ ID NO:5.
 3. An isolated hostcell transfected with the vector of claim
 2. 4. An isolatedpolynucleotide selected from the group consisting of SEQ ID NO: 14 andSEQ ID NO:
 3. 5. An isolated polynucleotide selected from the groupconsisting of SEQ ID NO: 15 and SEQ ID NO:
 4. 6. An isolated nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of: a) TAAGACGACG GCCTCAGACT TCTTGGGTAT TTGGACCACT GCACTGAAGAGATCATCTCT CCAGATTACT TTCCCCTGAG CTCCAGGCAC CATGAACTTT CCTTCTACAAAGGTTCCCTG GGCCGCCGTG ACGCTGCTGC TGCTGCTACT GCTGCCACCG GCGCTGCTGTCGCTTGGGGT GGACGCACAG CCTCTGCCCG ACTGCTGTCG CCAGAAGACG TGTTCCTGCCGTCTCTACGA ACTGTTGCAC GGAGCTGGCA ACCACGCTGC GGGTATCCTG ACTCTGGGAAAGCGGCGGCC TGGACCTCCA GGCCTCCAGG GACGGCTGCA GCGCCTCCTT CAGGCCAACGGTAACCACGC AGCTGGCATC CTGACCATGG GCCGCCGCGC AGGCGCAGAG CTAGAGCCACATCCCTGCTC TGGTCGCGGC TGTCCGACCG TAACTATCAC CGCTTTAGCA CCCCGGGGAGGGTCCGGAGT TTGAACCCAT CTTCTATCCT TGTCCTGATC CAAACTTCCC CCTCTGCTCGCCGCTGTCAG TCTCTTGGTA AATGGCAATA AAGACGTTTC TCTGTTGGTG TG (SEQ ID NO:4); and b) the complement of SEQ ID NO: 4.